JP6760680B2 - Method for Producing Metal-Containing Cluster Using Metallized Peptide - Google Patents

Description

The present invention relates to a method for producing a metal-containing cluster, particularly a metal-containing nanocluster, and a metal-containing sub-nanocluster.

As a method for producing nanoscale fine particles that can also be used as a catalyst, a method using a phenylazomethine dendrimer as a template, which is a unique method by the present inventors, has been energetically searched for and developed (Patent Document 1). The phenylazomethine dendrimer, which was developed by the present inventors and is being studied, has a large number of imine sites showing high coordination in the skeleton, and thus can be complexed with Lewis acid. The phenylazomethine dendrimer has an electron density gradient in which the electron density of imine increases from the terminal imine toward the center by continuously adding the electron donation from the imine from the terminal to the center. .. Due to this electron density gradient, when a metal salt is complexed with the phenylazomethine dendrimer, a stepwise complex formation occurs in which the innermost imine is preferentially complexed with the metal salt. By utilizing the stepwise complex forming ability of the phenylazomethine dendrimer, a binary system using two metal salts that accumulates different metal species in each layer, including a single one system such as platinum salt, etc. Furthermore, it is possible to accumulate precision heterometals of multiple types such as 3 types, 4 types, and 5 types, and it is possible to prepare a dendrimer complex in which the number and composition of metal salts are specified. By reducing the dendrimer complex coordinated with this metal salt, it becomes possible to form precisely controlled metal-containing sub-nanoclusters having an extremely small particle size distribution (Patent Documents 2 to 5).

Research by the present inventors has revealed that the physical and chemical properties of a metal-containing sub-nanocluster having about a dozen metal atoms largely depend on the number of constituent atoms and the type (). Non-Patent Documents 1 and 2). From this point of view, a synthesis method that defines the number of constituent atoms and their types is desired. However, since the method using the phenylazomethine dendrimer as a template accumulates depending on the complex formation constant of the metal, there is a limit in forming a sub-nanocluster by designing the number and composition of the metal in more detail.

Non-Patent Document 3 reports the synthesis of a metallized peptide in which a metal is bound to a plurality of amino acid residues. However, from the viewpoint of synthesizing metal-containing clusters such as nanoclusters, there are no known examples focusing on metallized peptides.

Japanese Patent No. 4511125 Japanese Unexamined Patent Publication No. 2010-18610 Japanese Unexamined Patent Publication No. 2013-159588 Japanese Unexamined Patent Publication No. 2007-23166 JP-A-2018-145172

K. Yamamoto et al. Angew. Chem. Int. Ed. 2015, 54, 9810-9815. K. Yamamoto et al. Sci. Adv. 2017, 3, e1700101. Chem. Lett. 2014, 43, 1167-1169.

Further, a fully automatic synthesis method capable of freely controlling the elements of the metal-containing nanocluster / nanoparticle catalyst is desired. An automatic synthesis method for peptides based on the Merryfield solid-phase synthesis method has been established, and an apparatus is commercially available. It is considered that the automatic synthesis of metal-containing nanocluster / nanoparticle catalysts will be possible by using peptides as templates.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel method for synthesizing a metal-containing cluster such as a nanocluster having a high degree of freedom in the number and composition of metal atoms. There is.
Another issue is to provide a method that contributes to speeding up catalyst search by enabling automatic synthesis.

As a result of diligent studies to solve the above problems, the present inventors have made a metallized peptide containing a plurality of the same or different metal atoms using the peptide as a template, whereby the number and composition of metal atoms can be freely adjusted. We have found that it is possible to synthesize monodisperse clusters with a high degree of degree, and have completed the present invention.

That is, in the method for producing a metal-containing cluster of the present invention, a metallized peptide containing the plurality of metal atoms in which the same or different metal atoms are bonded to a plurality of the same or different amino acid residues is reduced or hydrolyzed. It is characterized by forming a metal-containing cluster composed of the plurality of metal atoms and / or a metal oxide derived from the plurality of metal atoms by decomposing or chemically converting according to these.
Further, the method for producing a metal-containing cluster of the present invention is characterized in that the metallized peptide is synthesized by a solid-phase synthesis method.

According to the present invention, there is provided a novel method for synthesizing metal-containing clusters such as nanoclusters and sub-nanoclusters, which have a high degree of freedom in the number and composition of metal atoms.
A method for automatically synthesizing a metallized peptide by a solid-phase synthesis method is also provided.

(A) is the MALDI-TOF-MS spectrum of PtL-NH ₂ synthesized in the examples, and (b) is the ¹ H NMR spectrum thereof. It is a MALDI-TOF-MS spectrum of Fmoc-GluPtL-OAll synthesized in an example. It is a MALDI-TOF-MS spectrum of Fmoc- (Glu-PtL) ₄ -OAll synthesized in Examples ((a) is before purification, (b) is after purification). It is a complex model (a) of Fmoc- (Glu-PtL) _4- OAll / KB (0.05 wt%) and a HADDF-STEM image (b). The peptide was supported on KB using THF as a solvent. HADDF-STEM image of Pt ₄ cluster / KB. HADDF-STEM image of Fmoc- (Glu-PtL) _4- OAll / KB (0.05 wt%). The peptide was supported on KB using DMF as a solvent. HADDF-STEM image of Fmoc- (Glu-PtL) _4- OAll / KB (0.05 wt%). The peptide was supported on KB using CH ₃ Cl as a solvent. It is an XPS spectrum before and after firing of Fmoc- (Glu-PtL) ₄ -OAll ((a) is before firing, (b) is after firing). It is the XPS spectrum after calcination of Fmoc- (Glu-PtL) ₄ -Oall measured by changing the calcination time. The results before firing are also shown. It is an XPS spectrum after calcination of Fmoc- (Glu-PtL) ₄ -Oall measured by changing the calcination temperature. The results before firing are also shown. (A) is the MALDI-TOF-MS spectrum (matrix: 4-nitroaniline) of Fmoc- (E-NH-Pt) -COOH synthesized in the example, and (b) is the HPLC analysis result (detector: 214 nm). is there. It is the HPLC analysis result (detector: 214 nm) of NH _2- (E-NH-Pt) -COOH synthesized in the Example. (A) is the result of HPLC analysis (detector: 214 nm) of NH _2- (E-NH-Pt) _2- CONH ₂ synthesized in the examples, and (b) is the MALDI-TOF-MS spectrum (matrix) of the crude product. : CHCA). It is the HPLC analysis result (detector: 214 nm) of NH _2- (E-NH-Pt) _4- COOH synthesized in the Example. This is the result of comparing NH _2- (E-NH-Pt) _n- COOH (n = 1-4,6) synthesized in Examples under the same analytical HPLC conditions (detector: 214 nm). It is the HPLC analysis result (detector: 214 nm) of NH _2- (E-NH-Pt) _12- COOH synthesized in the Example. (A) is the result of HPLC analysis of NH _2- (E-NH-Pt) _3- CONH ₂ purified by PREP HPLC (detector: 214 nm), and (b) is the purified NH _2- (E) using each matrix. -NH-Pt) _12- COOH MALDI-TOF-MS spectrum (matrix: DCTB, CHCA, 4-nitroaniline, ditranol). Pt ₁₂ peptide / KB (Ketchen Black) 5 wt% atomic resolution HAADF-STEM image. (A) is an image observed in a magnified field of Pt ₁₂ peptide / KB 5 wt%, (b) is a molecular model of Pt ₁₂ peptide drawn by Gaussian View, and (c) is a molecular model of Pt ₁₂ peptide drawn by Gaussian View. The position of platinum. (A) is an atomic resolution HAADF-STEM image of Pt ₁₂ sub-nanoparticles / KB 2 wt%, (b) is a Pt ₁₂ sub-nanoparticle whose number of atoms is accurately counted, and (c) is an observed sub-nanoparticle size distribution. .. (A) is NH ₂ synthesized in Example - (E-NH-Pt) 19 -NH 2 in HPLC analysis (detector: 214 nm), (b) the crude product MALDI-TOF-MS spectrum ( Matrix: DCTB). Atomic resolution HAADF-STEM image of Pt ₁₉ sub-nanoparticles / KB 0.47 wt% @ Pt, where (a) is the overall field of view [1] scale bar: 5 nm (b) another overall field of view [2] scale. Bar: 10 nm. It is the total yield calculated from the total yield of each product in the catalytic reaction of toluene oxidation in the examples. All yields were evaluated based on an estimated metal support of 0.47 wt%. (A) is a ¹ H NMR spectrum (400 MHz, DMSO-d ₆ ) of Fmoc- (E-NH-pyL) -OAll synthesized in Examples, and (b) is a MALDI-TOF-MS spectrum (matrix: DCTB). is there. It is the HPLC analysis result (detector: 214 nm) of NH _2- (E-NH- (pyL-Ir))-COOH synthesized in the Example. It is the HPLC analysis result (detector: 214 nm) of NH _2- (E-NH- (pyL-Ir))-CONH ₂ after cutting from the resin. NH ₂ synthesized in Example - (E-NH-Pt) 3 - (E-NH- (pyL-Ir)) 3 -CONH 2 of HPLC analysis (detector: 214 nm) is. NH _2- (E-NH-Pt) _3- (E-NH- (pyL-Ir)) ₃ -CONH ₂ XPS spectra before and after calcination. Pt ₃ Ir ₃ sub-nanoparticles / KB 2 wt% atomic resolution HAADF-STEM image, (a) is a wide-field image, and (b) is a Pt ₃ Ir ₃ sub-nanoparticle of about 0.6 nm. (A) is an EDS mapping of Pt ₃ Ir ₃ subparticles, and (b) is an EDS spectrum of an element in the field of view.

The present invention will be described in detail below.
(Metalated peptide)
The metallized peptide (sometimes referred to as a metallopeptide) used in the present invention includes the plurality of identical or different metal atoms in which a metal atom is bonded to a plurality of the same or different amino acid residues.

The form in which a plurality of metal atoms are bonded to a plurality of amino acid residues includes a form in which metal atoms are complex-formed, ionic bonded, or covalently bonded. In a typical example, a metal atom binds to multiple amino acid residues by complex formation.

The metal atom is not particularly limited, and for example, titanium, vanadium, iron, cobalt, nickel, copper, zinc, gallium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, indium, tin, antimony, hafnium, etc. Examples thereof include tantalum, tungsten, ruthenium, osmium, iridium, platinum, gold, mercury and bismuth. These may be used individually by 1 type, and may be used in combination of 2 or more type. When two or more are used in combination, alloy clusters can be formed.

The metalated peptide contains, for example, the structure of the following formula (I).

(Wherein, R ¹ represents an amino acid residue, n may be different in each identical .n number of R ¹ is showing an integer of 2 or more, at least two or more amino acids of the n R ¹ A metal atom is attached to the residue.)

The building blocks of metalated peptides are amino acids and / or amino acid derivatives. The metalated peptide may be an oligomer or polymer of such building blocks. Here, the oligomer refers to an oligopeptide in which approximately 10 or less amino acid residues are bound by peptide bonds. A polymer is a polypeptide in which approximately 11 or more amino acid residues are bound by peptide bonds.

In the present invention, the "amino acid" refers to a compound having an amino group (-NH ₂ ) and a carboxy group in the molecule, and as defined by a general amino acid, hydrogen of the amino group is associated with other parts in the molecule. It also includes imino acid, which is a cyclic compound that has been substituted to become a secondary amine.

In the present invention, the "amino acid derivative" refers to a compound having a structure in which any group of the above amino acids is modified or substituted. For example, naturally occurring amino acid derivatives, which are components that construct a living body, can be mentioned.

The number of amino acid residues in the metallized peptide is not particularly limited as long as it is 2 or more, and is, for example, 100 or less. From the viewpoint of synthesizing nanoclusters, the number of amino acid residues bonded to metal atoms is preferably 3 or more, and more preferably 4 or more. Further, 60 or less is preferable, and 50 or less is more preferable. In a typical example, all amino acid residues in a metalated peptide are amino acid residues that bind to metal atoms.

In the present invention, the metallized peptide is not particularly limited as long as it has a structural unit of an amino acid and / or an amino acid derivative. For example, the terminal may be any organic group or the like as long as the effect of the present invention is not impaired, and may be an appropriate one depending on the convenience of the synthetic route and the like.

The amino acid from which the metallized peptide is derived is not particularly limited, and examples thereof include 20 kinds of α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids constituting natural proteins.

From the viewpoint of having an amino acid residue that binds to a metal atom, the amino acid and the amino acid derivative from which the metallized peptide is derived preferably have a reactive functional group at the residue. This reactive functional group reacts with the reactive functional group of the metal salt compound to form an amino acid residue that binds to a metal atom. Examples of the reactive functional group include an amino group, a hydroxyl group, a carboxyl group, a thiol group, a phosphonic acid group and the like. Examples of amino acids having such a reactive functional group as residues include arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, proline, serine, threonine, tryptophan, and tyrosine. Examples of the amino acid derivative having a reactive functional group as a residue include creatine, ornithine, hydroxyproline, hydroxylysine, phosphosellin, theanine, kainic acid, and triricholomic acid.

The structure in which a metal atom is bonded to an amino acid residue in a metallized peptide is, for example, a structure in which the metal salt compound (A) is bound to the amino acid residue (B) by a reaction. Examples of the reaction include a condensation reaction using the above-mentioned reactive functional groups, particularly an amidation reaction and an esterification reaction.

Among these, from the viewpoint of suppressing the decomposition of the bond due to hydrolysis when deprotecting with a base, the structure in which the metal atom is bonded to the amino acid residue is the metal salt compound (A) and the amino acid residue. It is preferable that these are bonded by an amidation reaction with the group (B). The bond, which is the same bond as the peptide in the main chain, improves stability. In this case, for example, one of the metal salt compound (A) and the amino acid residue (B) has an amino group, and the other has a carboxyl group.

The metal salt compound (A) is represented by, for example, MR ² . Here, M indicates a metal and R ² indicates the chemical structure of the residue of the metal atom M. R ² may be in the singular or plural combinations, depending on the valence of M. The mass of the entire R ² is not particularly limited, but may be, for example, 400 or less, 300 or less, or 200 or less in terms of atomic weight.

The metal salt compound (A) has a broad meaning including a salt of a metal and an anion, a complex of a ligand, and the like. The counter anion or ligand in the metal salt compound is not particularly limited, but is, for example, halogen ions such as chloride ion, bromide ion, and iodide ion, trifluoromethanesulfonic acid, acetic acid, acetylacetone, salen, and cyclopentadi. Examples include enyl. In addition to these, an organic group having a functional group that reacts with the above-mentioned reactive functional group at the amino acid residue (B) is preferably contained.

Preferred examples of the metal salt compound (A) include those represented by the following formulas (II) and (III).

(In the formula, ^{Ra to} R ^e indicate a hydrogen atom or a substituent, X indicates the chemical structure of the residue of the metal atom M, and ^RA indicates a group containing a functional group capable of forming an amide bond or an ester bond. Show.)

(In the formula, R ^{f to} R ^l indicate a hydrogen atom or a substituent, X indicates the chemical structure of the residue of the metal atom M, and ^RA indicates a group containing a functional group capable of forming an amide bond or an ester bond. Show.)

When R ^{a to} R ^l are substituents, the substituent is not particularly limited, but for example, a halogen atom such as a chlorine atom, a bromine atom or a fluorine atom, or an alkyl group such as a methyl group or an ethyl group (for example, the number of carbon atoms). 1-6), heteroelement-containing groups (hydroxy group, amino group, nitro group, thiol group, etc.) whose bond sites are heteroelements and do not contain carbon, halogen atoms, oxygen-containing groups (hydroxy group, oxo group, ether group, etc.) , Carboxyl group, aldehyde group, etc.), nitrogen-containing group (cyano group, primary to tertiary amino group, nitro group, etc.), sulfur-containing group, phosphorus-containing group, etc. And an alkyl group in which the CC bond is interrupted (for example, 1 to 6 carbon atoms) and the like. These substituents may have one or more. Among them, the number of substituents of ^{Ra to} R ^l is preferably one or less, and more preferably no substituents.

Examples of X include counter anions and ligands.

^RA is not particularly limited, but is, for example, a hydrocarbon group (for example, 1 to 20 carbon atoms) containing a functional group capable of forming an amide bond or an ester bond as a substituent, for example, an aliphatic hydrocarbon group or an aromatic hydrocarbon. Groups, alicyclic hydrocarbon groups and the like can be mentioned.

As described above, the functional group of ^RA is preferably a group that forms an amide bond from the viewpoint of suppressing the decomposition of the bond due to hydrolysis when deprotecting with a base. When the reactive functional group at the amino acid residue (B) is a carboxyl group, an amino group is preferable.

In the above formula (II), a cyclic structure is formed between the imine nitrogen atom forming the azomethine bond in the phenylazomethine structure, the carbon atom, the phenyl ring bonded to the carbon atom, and the metal atom M of the metal salt compound. are doing. In this cyclic structure, metal atoms form a strong bond, and the bond stability of the metal atom is excellent. In the above formula (III), a cyclic structure is similarly formed by a structure containing pyridine. This cyclic structure does not decompose the imine site and can be used in an aqueous system, and has excellent resistance to strong acids.

In the present invention, the metallized peptide is used for liquid phase synthesis in which a metal salt compound (A) is reacted with an amino acid residue (B) after synthesizing a peptide, or an amino acid in a metal salt compound (A) and an amino acid or a derivative thereof. It is produced by a solid-phase synthesis method in which these are bound by a reaction with the residue (B), a metallized amino acid or a derivative thereof is synthesized in advance, and then sequentially synthesized.

The liquid-Phase Peptide Synthesis method has the advantage that the reaction points can be modified at one time and can be easily synthesized.

Since the solid-phase synthesis method (Solid-Phase Peptide Synthesis) enables sequential synthesis, there is a degree of freedom in the composition of metal clusters. It can be synthesized in a high yield and in a short time, and even if it is a long chain, it is possible to suppress a decrease in reactivity such as racemization and reaction sites that are unlikely to appear. According to the solid-phase synthesis method, a multi-element polynuclear complex sequence can be synthesized and reduced as a template to synthesize a multi-element cluster having size selectivity and a free composition. That is, by performing step-by-step, it has a great advantage that peptides in which various metals and sequences are programmed can be synthesized, and a peptide template in which the length of the chain is extended can be designed with a free composition. .. The peptide solid phase synthesis method has been applied to combinatorial chemistry, automatic synthesis, virtual libraries, etc., and is a proven method in terms of free composition of templates.

In an example of the solid-phase synthesis method, beads or the like of a polystyrene polymer gel whose surface is modified with an amino group are used as a solid phase, and metallized amino acid chains are extended one by one by a dehydration reaction. When the sequence of the target peptide is completed, it is excised from the solid-phase surface to obtain the target substance. In the solid-phase synthesis method, synthesis proceeds from the C-terminal side to the N-terminal side. Therefore, the C-terminal amino acid is fixed on the carrier and the synthesis is started. Fmoc groups, Boc groups and the like are used to protect amino groups in order to prevent amino acids from self-condensing to form diketopiperazines or reacting with peptides after the formation of oligomers. Further, for the amide bond forming reaction, a uronium-based condensing agent such as HATU or HBTU, a combination of 1-hydroxybenzotriazole and N, N'-dicyclohexylcarbodiimide, a carbodiimide-based condensing agent such as diisopropylcarbodiimide, or the like should be used as the condensing agent. Can be done. When the reaction between the surface of the solid phase and the metallized amino acids is completed, the solid phase is washed with a solvent to remove residual amino acids and the like. After that, when the protecting group of the metallized amino acid bonded to the solid phase is removed (deprotected), the amino group serving as the next reaction point reappears on the surface of the solid phase. By repeating this procedure while appropriately changing the metalated amino acid to be used, a metalated peptide having the desired sequence can be synthesized with high accuracy. An automatic synthesizer that automates this procedure is also available.

The flow of solid-phase synthesis will be described by taking a 12-residue platinum peptide as an example. In this example, the Pt unit (platinum complex-bonded amino acid) for synthesizing the metallized peptide has a structure represented by the formula (II), and contains the metal salt compound (A) and the amino acid residue (B). These are combined by the reaction of. The flow from solid-phase synthesis to the synthesis of 12-atom Pt sub-nanoparticles is roughly as follows.
(1) Synthesize a metal complex that connects to the side chain of the peptide, and then synthesize an artificial amino acid that is bound to glutamic acid.
(2) Subsequently, the synthesized amino acid is condensed by a solid phase synthesis method and cut out from the resin to obtain a 12-residue platinum peptide.
(3) Finally, the corresponding number of platinum sub-nanoparticles are synthesized by reduction treatment.
Similar to the method for synthesizing sub-nanoparticles using a dendrimer, a 12-residue platinum peptide may be synthesized in solid phase, supported on a carrier and then reduced to synthesize platinum sub-nanoparticles, or a 12-residue platinum peptide may be synthesized. May be solid-phase synthesized, and platinum sub-nanoparticles may be synthesized by reduction treatment and then supported on a carrier. In the synthesis of sub-nanoparticles with a dendrimer, it has been confirmed that aggregation can be suppressed by reducing after complex formation and then impregnating with a PVP (polyvinylpyrrolidone) solution. For example, metallized peptides are reduced after synthesis. It is then considered that the PVP solution is impregnated to prevent aggregation and then supported on a carrier.

In the synthesis scheme of the side chain-modified Pt complex in (1), a ligand having an imine bond is quantitatively synthesized using ethylamine in which benzaldehyde and one-sided amine are protected as a starting material. Further, a platinum salt and triphenylphosphine are added to the synthesized ligand to synthesize a platinum cyclometallate complex, and the terminal is deprotected with a strong acid to synthesize an amine platinum complex for peptide side chain modification. In the synthesis scheme of Pt units (platinum complex-bonded amino acids), the above-synthesized amine platinum complex is modified into a side chain using glutamic acid whose N-terminal and C-terminal are protected by protecting groups. Then, one protecting group is removed with tetrakis palladium to synthesize a platinum complex-bonded amino acid applicable to solid-phase synthesis.

In (2), as STEP1, the solid phase and the Pt unit are condensed to chemically bond the platinum peptide to the solid phase. Then, piperidine is used as STEP2 to remove the protecting group at the tip of the peptide to make it a terminal amine, and a platinum unit is added as STEP3 and condensed to synthesize a platinum peptide extended by 1 unit. STEP2 and STEP3 are repeated to extend the platinum peptide to an arbitrary number, and then the peptide is treated with an acid in STEP4 to synthesize a peptide having a desired platinum number.

In the solid-phase synthesis of metallized peptides as described above, the condition for final excision is preferably a weak acid rather than a strong acid in terms of suppressing the decomposition of the complex. When a weak acid is used, a solid phase having a linker that can be cut out with the weak acid is used. Further, if unreacted reaction points remain during each condensation, 11 or 10 residues will be mixed even if the condensation is repeated 12 times. In terms of increasing the condensation efficiency, a uronium-based condensing agent is preferable, and the synthetic purity can be increased as compared with a carbodiimide-based condensing agent.

The example below shows the solid phase synthesis cycle of an alloy. Together with the Pt unit, the side chain complex is bound to the metal salt compound (A) using an Ir complex having a pyridine-type structure represented by the formula (III) by the reaction with the amino acid residue (B). Use the Ir unit. The Pt ₃ Ir ₃ metallopeptide is synthesized by extending the Pt unit three times and then extending the Ir unit three times.

In the method for producing a metal-containing cluster of the present invention, a metallized peptide containing the plurality of metal atoms in which the same or different metal atoms are bonded to a plurality of the same or different amino acid residues is reduced or hydrolyzed. Alternatively, or by chemically converting according to these, a metal-containing cluster composed of the plurality of metal atoms and / or metal oxides derived thereto is formed.
In the present invention, the metallized peptide can be supported on the surface of the carrier and then reduced or hydrolyzed, or chemically converted according to these to form the metal-containing cluster on the surface of the carrier. it can.
Alternatively, the metallized peptide is not supported on a carrier, but is reduced or hydrolyzed, or chemically converted in accordance with these, thereby resulting from the plurality of identical or different metal atoms and / or the same. It is also possible to form metal-containing clusters composed of metal oxides. For example, in the synthesis of sub-nanoparticles with a dendrimer, it has been confirmed that aggregation is suppressed by reducing after complex formation and then impregnating with a PVP solution. For example, metallized peptides are reduced after synthesis and then PVP. It is considered that the solution is impregnated to prevent aggregation and then supported on a carrier.

(Support and reduction on carrier)
The plurality of metal atoms and / or the plurality of metal atoms and / or the plurality of metal atoms and / or by carrying a metallized peptide containing a plurality of the same or different metals in which a metal is bonded to an amino acid residue and then reducing or hydrolyzing the carrier surface A metal-containing cluster made of a metal oxide derived from this can be formed. After reduction, the metal becomes a metal oxide by oxidation or opening to the atmosphere, and a metal-containing cluster composed of the plurality of metal oxides can be formed on the surface of the carrier.

By supporting the metallized peptide on a carrier, aggregation of metal particles and the like can be suppressed. The shape of the carrier is not particularly limited, and may be various, such as granular, fibrous, granular, film-like, and plate-like. Considering that the surface area per unit weight is large, granular (powdered) is preferable.

As the carrier, carbon materials such as carbon black (Ketjen black, oil furnace black, gas black, acetylene black, lamp black, thermal black, channel black, etc.), activated carbon, carbon fiber and other amorphous (microcrystals) Examples thereof include nanocarbons such as carbon, fullerene, nanotubes, graphene and graphene oxide, three-dimensional crystals such as graphite, and graphitized mesoporous carbon. These carbon materials may be porous materials and can support single element clusters on the pore surface.

In addition, an inorganic material can be used as the carrier. Examples of the inorganic material include silica gel, alumina, titania, magnesia, zirconia, iron oxide, copper oxide, glass, silica sand, talc, mica, clay, and wallusnite.

Preferred carriers in terms of carrier internal area, affinity, versatility, stability and durability include Ketjenblack (KB), graphitized mesoporous carbon (GMC), graphene oxide (GO) and aluminum oxide (Al ₂ O). ₃ ) etc. can be mentioned.

The support on the carrier is carried out, for example, by using a solution dissolved in an appropriate solvent such as an organic solvent, contacting the carrier by mixing, impregnating, coating, dropping or the like with a carrier dispersion, and then drying. Can be done. If necessary, a pulverization treatment may be performed.

As the supported amount on the carrier, it is considered to select an appropriate supported amount at which the complex does not aggregate in order to achieve the metal cluster synthesis in which the number of atoms is controlled. If the metallized peptide before calcination is supported at a low density, aggregation due to calcination is unlikely to occur. Further, as the number of atoms in the metal cluster decreases, the number of particles per area increases and the average spacing between the particles becomes narrower, so that aggregation is likely to occur. Therefore, it is desirable that the amount supported is smaller in terms of suppressing aggregation. The balance between the large amount of support for the purpose of use such as catalytic activity and the requirement that aggregation is unlikely to occur as the performance of the purpose of use is such that the amount of support is large so that the metallized peptide does not aggregate. Is considered to be selected.

The reduction can be carried out in the gas phase or in a solution using a reducing agent that has a reducing action on the metal of the metallized peptide and can reduce this to a zero-valent state.

In the case of reduction in the gas phase, the carrier is supported on a carrier and then calcined in a mixed gas atmosphere with hydrogen gas or hydrogen-containing nitrogen or argon. By supporting the metallized peptide on a carrier, clusters can be formed without aggregation.

The calcination temperature is not particularly limited depending on the type of metallized peptide, etc., but in consideration of the progress of reduction by calcination, the progress calcination time not becoming too long, the suppression of cluster aggregation, etc., for example, 200 to 300 ° C. is considered. The firing time is not particularly limited, but for example, 1 to 24 hours are considered.

When reducing in a solution, the reducing agent includes, for example, sodium borohydride, sodium borohydride, hydrogen, hydrazines, lithium aluminum borohydride, diisobutyl aluminum hydride, lithium borohydride, tetra n borohydride. -Butylammonium, boron methylammonium hydride, lithium triethylborohydride, borane complexes, sodium triacetoxyborane, zinc borohydride, lithium tributylboron hydride, potassium tributylboron hydride, Schwartz reagent, Stryker reagent, hydrogenation Examples thereof include tributyltin, sodium borohydride, lithium borohydride, calcium hydride, benzophenone ketyl radicals, metal naphthalenides, hydrogen peroxide and the like.

By reducing the metallized peptide in this way, a metal cluster having a size corresponding to the number of metal atoms of the metallized peptide can be synthesized.

In the case of titanium oxide, for example, hydrolysis results in titanium hydroxide by hydrolysis, and metal clusters are formed by firing.

The method of the present invention is suitable for synthesizing metal nanoparticles among metal-containing clusters. A polyhedral atomic group formed by aggregating several atoms and directly bonding a part or all of them is generally called a cluster, and in that sense, metal nanoparticles are clusters.

The metal-containing nanoparticles such as metal nanoparticles obtained by the method of the present invention are expected to be applied as materials for various fields including catalysts, for example, pharmaceuticals, electronic functional materials, environment-friendly materials, luminescent materials, etc. Will be done.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
Synthesis of PtL-NH ₂ A platinum complex having an amine introduced at the end was synthesized as a raw material complex by the route shown below.

Specifically, aminoethane-Boc (2.00 g) and distilled benzaldehyde (1.34 g) are stirred in a toluene solvent at 115 ° C. for 24 hours to distill off the solvent to collect the ligand (Ligand-Boc). It was synthesized at a rate of 92%.

Then, 1.21 g of Ligand-Boc and 2.01 g of K ₂ [PtCl ₄ ] were refluxed in an MeOH solvent at 70 ° C. for 24 hours, returned to room temperature, 1.26 g of PPh ₃ was added, and the mixture was stirred for 3 hours, and then silica gel. By developing and purifying with a developing solvent of ethyl acetate: hexane = 1: 1 using column chromatography, a platinum complex (PtL-Boc) was obtained in a yield of 0.40 g and a yield of 11.2%. The Boc group is deprotected by stirring PtL-Boc in a CH ₂ Cl ₂ / TFA 50% solution at room temperature for 10 minutes, and purified by silica gel column chromatography while applying a gradient with ethyl acetate: methanol solvent. The target product, PtL-NH _2, was obtained in a yield of 98%. Identification was performed by ¹ H NMR and MALDI-TOF-MS (FIGS. 1 (a) and 1 (b)).

Synthesis of Fmoc-GluPtL-NH-OAll As shown below, a condensing agent was added using PtL-NH ₂ synthesized with Fmoc-Glu-OAll as a raw material, and the condensation reaction was carried out by stirring at room temperature.

When the peak of the raw material was followed by MALDI-TOF-MS after the start of the reaction, it was confirmed whether the peak of PtL-NH ₂ disappeared 3 hours after the start of the reaction. Then, after liquid separation treatment was performed to remove the condensing agent, purification was performed by silica gel column chromatography using ethyl acetate: hexane = 1: 1 as a developing solvent (yield 55%). The obtained yellow oil was identified by MALDI-TOF-MS (Fig. 2).

Synthesis of Fmoc- (Glu-PtL) _4- OAll An amine complex was introduced into the carboxylic acid side chain of the peptide by the route shown below to synthesize a tetranuclear metallopeptide.

Specifically, Fmoc-Glu _4- OAll (5.0 mg) and PtL-NH ₂ (19.4 mg) were dissolved in a DMF solvent under an Ar atmosphere, a condensing agent was added, and the mixture was stirred at room temperature for 4 days for synthesis. .. After that, the condensing agent was removed by liquid separation treatment, supported on silica gel, and purified by using flash column chromatography with CHCl ₃ : MeOH while applying a gradient to obtain the desired product (m / z = 3253) in yield 18. Obtained in%. Identification was performed by MALDI-TOF-MS (Fig. 3).

Next, STEM observation of the obtained metallopeptide cluster was performed.
Direct observation of KB / Fmoc-(Glu-PtL) ₄ -OAll 0.05% wt
1 mg of Fmoc- (Glu-PtL) ₄ -OAll was dissolved in 100 ml of THF to prepare a 3 μM solution, and 20 mg of acid-treated KB (Ketjen black) was added and supported while stirring in an eggplant flask. The supported KB was filtered through a membrane filter, taken out, dried, dispersed in MeOH, and dropped cast into Cu grit 3 μl × 3 times to prepare a TEM sample. When the complex was directly observed using HAADF-STEM, the presence of four platinums thought to be derived from tetranuclear metallopeptide could be confirmed as shown in FIG. 4 (b). From the complex model construction in FIG. 4 (a), the size of the tetranuclear complex is predicted to be approximately 2 to 3 nm, suggesting that a platinum complex matching the size may have been synthesized.

Direct observation of KB / Pt ₄ clusters Using the KB / Fmoc- (Glu-PtL) _4- OAll 0.05% wt prepared above, 250 ° C in a hydrogen reduction furnace under a mixed gas of H ₂ / N ₂ 3%. It was fired under the condition of 18 hours. The calcined KB was dispersed in MeOH in the same manner as above, dropped cast into Cu grit 3 μl × 3 times, a TEM sample was prepared, and direct observation was performed with HADDF-STEM. As shown in Fig. 5, we also succeeded in confirming a substance that seems to be a Pt ₄ cluster.

Examination of supported solvent for Fmoc- (Glu-PtL) ₄ -OAll When the supported KB in which the supported solvent was changed to DMF and CHCl ₃ was observed, the dispersed state as shown in FIGS. 6 and 7 was observed. As can be seen by comparing the two, even if the same amount of Fmoc- (Glu-PtL) ₄ -O All is added, the amount supported on the KB is significantly different. Comparing the results including the THF result of (1), the amount of KB supported by the solvent was DMF>THF> CH ₃ Cl. From this result, it was found that the solvent having a higher polarity has a stronger interaction with KB and is a solvent that easily supports a carrier. In terms of supporting the metallopeptide in an isolated dispersion, the CH ₃ Cl condition is optimal under the 0.05 wt% condition in consideration of dispersibility.

XPS measurement before and after calcination of metallopeptide
Fmoc- (Glu-PtL) ₄ -OAll 3.8 μM solution was drop-cast 3 ml × 3 times on the HOPG substrate, and then XPS measurement was performed to measure the charge state of Fmoc- (Glu-PtL) ₄ -OAll. .. The measured sample was further calcined at 250 ° C. for 18 hours under a H ₂ / N ₂ 3% atmosphere, and then the same sample was measured again by XPS. The result is shown in FIG. The binding energies of the Pt 4f orbits before firing at 4f _7/2 and 4f _5/2 were 72.8eV and 75.8eV, respectively, and the orbits peculiar to Pt ²⁺ could be measured. On the other hand, the binding energies of the Pt 4f orbital 4f _7/2 and 4f _5/2 after calcination are 71.7 eV and 74.9 eV, respectively, and the peak of the binding energy shifts to the reduction side by about 1 eV compared to before calcination. It was confirmed that Pt ²⁺ was reduced to Pt ⁰ .

Firing time, calcination temperature The calcination time was changed from the above conditions, and the sample was measured by XPS. The result is shown in FIG. From the comparison with before calcination, the reduction by calcination was confirmed from 3 hours to 18 hours from the peak shift of the binding energy to the reduction side at 4f _7/2 and 4f _5/2 of the Pt 4f orbit.

The firing temperature was changed from the above conditions, and the sample was measured by XPS. The firing time was 3 hours. The result is as shown in FIG. From the comparison with before calcination, the reduction by calcination was confirmed from the peak shift of the binding energy to the reduction side at 4f _7/2 and 4f _5/2 of the Pt 4f orbital from 225 ° C to 250 ° C.

Next, solid-phase synthesis of peptides was investigated. There are two methods for solid-phase synthesis of peptides: manual synthesis and automatic peptide synthesizer. In the following, peptide automatic synthesizer (manufactured by Biotage), HPLC (manufactured by Shimadzu), centrifuge (manufactured by Eppendorf) Was used.

Synthesis of solid-phase-based precursor Fmoc- (E-NH-Pt) -COOH

Add Fmoc- (E-NH-Pt) -OAll (2.18 g, 2.12 mmol), Pd (PPh ₃ ) (0.24 mg, 0.21 mmol) and dimedone (0.68 mg, 4.84 mmol) to a two-port eggplant flask (100 ml). Ar substitution in the system. A septum rubber was attached to one side, and a three-way cock was attached to the other side. Dehydrated CH ₂ Cl ₂ was added with a 20 ml syringe, and the inside of the system was replaced with Ar again. A balloon was attached to the tip of the three-way cock, and the mixture was stirred at room temperature for 3 hours while being kept under Ar. After stirring, the mixture was opened to the atmosphere and MALDI-TOF-MS was measured. As a result, the peak of the raw material, m / z = 996, disappeared, confirming the completion of the reaction. After distilling off the reaction solution under reduced pressure to concentrate the solution, the solution was directly charged on the upper part of the silica gel column by flash column chromatography, then flowed at 10 CV with CHCl ₃ , and then gradient at 10 CV until CHCl ₃ : MeOH = 90: 10. The target product was separated by flowing the developing solvent while applying the above solution. The solvent of the separated fraction was distilled off under reduced pressure and dried under vacuum for 1 day to obtain a yellow powder. The yield was 1.50 g and the yield was 71.8%, and identification was performed using MALDI-TOF-MS and analysis HPLC (FIGS. 11 (a) and 11 (b)).

Synthesis of Fmoc- (E-NH-Pt) _3- CONH ₂ by an automatic synthesizer The mononuclear Pt metallopeptide synthesized in this way with high purity was used as a precursor for synthesis. The flow of peptide synthesis is as follows: a peptide having an N-terminal protected with a solid phase is condensed, and the next peptide is added to the deprotected N-terminal again to condense the solid phase. When the peptide chain is extended while being bound and the target sequence is obtained, the target substance is obtained by cleaving from the solid phase using a Crivage solution such as an acid. Therefore, by using an automatic synthesizer, it is possible to program a fixed peptide residue and automatically perform a sequence.

Solid-phase synthesis of metallopeptides under weak acid excision conditions So far, it has been observed that platinum metallopeptides are partially decomposed by excision from the solid phase using a strong acid. Therefore, the resin used was changed from Rink Amid ChemMatrix resin, which is cleaved under the strong acid conditions used, to Trityl-OH ChemMatrix resin, which is used when synthesizing protective peptides. Since this Trityl-OH ChemMatrix resin has a hydroxy group at the end, it is necessary to convert the end to chloride and activate it before using the resin. Specifically, when using on a 0.01 mmol scale, before use, the resin (25 mg) is swollen by shaking with ₂ ml of CH ₂ Cl ₂ for 3 hours to discharge the solution, and then the SOICl ₂ / CH ₂ Cl ₂ 2% solution. 2 ml and shake overnight. Then, the resin was washed 5 times with 1 ml of CH ₂ Cl ₂ and 3 times with 1 ml of DIPEA / CH ₂ Cl ₂ 2% solution to chlorinate the ends. To further extend the peptide as it is, add Fmoc- (E-NH-Pt) -COOH (29.7 mg, 0.03 mmol) and DIPEA (5.17 mg, 0.04 mmol) to 1 ml of CH ₂ Cl ₂ overnight. A sequence in which one residue of the peptide was extended to the solid phase was synthesized by shaking, and the N-terminal was deprotected by reacting the N-terminal with piperidine / DMF 20% (v / v) for 15 min. After that, Crivage was performed from the solid phase using a cut-out cocktail of CH ₂ Cl ₂ : TFA: TIPS = 96.5: 1: 2.5 (v / v) to recover the solution, and reprecipitation was performed by adding 13 ml of tert-butyl methyl ether. did. After decanting the precipitated substance, 13 ml of diethyl ether was added for washing, and then the operation of precipitating with a centrifuge was repeated twice to obtain a yellow powder.

The resulting powder _{CH 3 CN / H 2 O 30} % dissolved in (0.1% TFA) _{solution, CH 3 CN / H 2 O} 95 in _{Analysis HPLC CH 3 CN / H 2} O30% from (0.1% TFA) A single main peak was observed when the analysis was performed while applying a gradient to% (0.1% TFA) (Fig. 12). From this result, the ratio of the main peak area to the total UV peak area was calculated to be 96.7%. Therefore, we succeeded in attaching and detaching the mononuclear Pt metallopeptide using Trityl-OH ChemMatrix resin. ..

Synthesis of NH _2- (E-NH-Pt) _2- CONH ₂ A one-residue Pt metallopeptide with deprotected ends was prepared by following the same procedure as above. Then, in order to further extend one residue, Fmoc- (E-NH-Pt) -COOH (29.7 mg, 0.03 mmol), COMU (17.1 mg, 0.04 mmol), and DIPEA (5.17 mg, 0.04 mmol) were added to 1 ml of DMF. Was added, the mixture was shaken for 1 hour, and then washed with DMF. Then, the N-terminus was deprotected by reacting the N-terminus with piperidine / DMF 20% (v / v) for 15 min. A solid phase was cut out and reprecipitated in the same manner as described above to obtain a yellow powder. Dissolve the powder in CH ₃ CN / H ₂ O 30% (0.1% TFA) solution and analyze HPLC from CH ₃ CN / H ₂ O 30% (0.1% TFA) to CH ₃ CN / H ₂ O 95% (0.1% TFA). When the same analysis was performed while applying the gradient to 0.1% TFA), the main peak could be observed at a longer time (24.4 min) of RT than when NH _2- (E-NH-Pt) ₁ -CONH ₂ . From this, it was suggested that when the residue was extended, the polarity of the molecule changed to low polarity (Fig. 13 (a)). The area ratio of the main peak to the total area ratio of the peak was 81.8%. When the crude powder was measured by MALDI-TOF-MS, a peak of (m / z = 1485), which seems to be NH _2- (E-NH-Pt) _2- CONH ₂ , was observed. Solid-phase synthesis of Pt metallopeptide was successful (Fig. 13 (b)).

Automatic synthesis using a resin under weak acid excision conditions In the same manner as described above, a 1-residue Pt metallopeptide having a deprotected terminal was prepared, and the second and subsequent residues were synthesized using an automatic synthesizer.

For elongation, HBTU / DMF 0.2M as a condensing agent, HOBt / DMF 0.2M as a condensation aid, N-ethyl-diisopropylamine / NMP 0.2M as an auxiliary base, and Fmoc- (E-NH-Pt) -COOH / as a condensing peptide. DMF 0.5M was added, and the procedure was performed under the conditions of microwave 75 ° C. and 5 min. The deprotection of the terminal Fmoc group was reacted with a piperidine / DMF 20% (v / v) solution. After performing the deprotection → elongation process three times, put in the final deprotection process to make the terminal amine and take out the 5 ml vial from the automatic synthesizer, CH ₂ Cl ₂ : TFA: TIPS = 96.5: 1: 2.5 A yellow powder of NH _2- (E-NH-Pt) _4- CONH ₂ was obtained by cribaging from the solid phase using a (v / v) cut-out cocktail, recovering the solution, and performing a reprecipitation operation.

The resulting powder _{CH 3 CN / H 2 O 30} % dissolved in (0.1% TFA) _{solution, CH 3 CN / H 2 O} 95 in _{Analysis HPLC CH 3 CN / H 2} O30% from (0.1% TFA) When the analysis was performed while applying a gradient to% (0.1% TFA), the main peak was observed at the position of RT = 31.5 min (Fig. 14). The area ratio of the main peak to the total peak area was 88%, and we succeeded in synthesizing Pt ₄ metallopeptide with high purity using an automatic synthesizer.

Similarly, when Pt ₃ metallopeptide synthesis or Pt ₆ metallopeptide synthesis is performed and the retention times are compared under the same HPLC conditions, the retention time gradually increases, that is, the polarity decreases as the number of residues increases. It was found that if it was about Pt ₆ metallopeptide, it was possible to search for metallopeptide synthesis conditions with high purity and target sequence even in the crude state (Fig. 15).

Synthesis of Pt ₁₂ metallopeptide In the same manner as above, prepare a 1-residue Pt metallopeptide whose terminal is deprotected, and deprotect the last N-terminal after repeating deprotection → extension 11 times under the same extension conditions. As a result, the synthesis was completed with the terminal as amine. Take out the 5 ml vial from the automatic synthesizer, cleave it from the solid phase using a cut-out cocktail of CH ₂ Cl ₂ : TFA: TIPS = 96.5: 1: 2.5 (v / v), collect the solution, and perform the reprecipitation operation. By doing so, a yellow powder of NH _2- (E-NH-Pt) _12- CONH ₂ was obtained. Unlike up to 6 residues, the resulting powder could not be dissolved in CH ₃ CN / H ₂ O 30% (0.1% TFA) solution, resulting in CH ₃ CN / H ₂ O 50% (0.1%). It was dissolved in% TFA) solution and analyzed by Analysis HPLC from CH ₃ CN / H ₂ O 50% (0.1% TFA) to CH ₃ CN / H ₂ O 95% (0.1% TFA). However, many peaks were observed (Fig. 16).

Assuming that this peak is due to poor elongation, the one with the longest reaction extension is considered to have the longest retention time, as shown in FIG. 12, so retention time = 25.2 min. The peak portion was recovered as the peak of NH _2- (E-NH-Pt) _12- COOH using a preparative column under the same conditions while applying a gradient. When the recovered solution was evaluated again by Analysis HPLC, it did not reach a single peak, but the excess peak could be removed and recovered (FIG. 17 (a)). By removing the solvent from the recovered solution with a freeze-dryer, 450 μg of a peptide thought to be NH _2- (E-NH-Pt) _12- COOH was recovered.
Figure 17 (b) compares the recovered metallopeptides in different matrices using MALDI-TOF-MS, with regular intervals (approximately m / z =) near m / z = 7000 to 9000 from the MS spectrum. Since the peak appears at 260), it is considered that PPh ₃ (Mw = 262) in the complex part of the metallopeptide is seen off by the laser of MALDI-TOF-MS. From this, the recovered metallopeptide can be synthesized as Pt ₁₂ metallopeptide based on the results of analytical HPLC and MALDI-TOF-MS.

STEM observation of Pt ₁₂ metallopeptide / KB 2 wt% Weigh the NH _2- (E-NH-Pt) _12- COOH thus synthesized with a 20 μg precision balance, add it to a 10 m vial, and then add 2 ml of CHCl ₃ . In addition, it was dissolved. Also, add 1 mg KB to another 10 ml vial, then add 2 ml CHCl ₃ and sonicate for 15 minutes to disperse. After the ultrasonic dispersion is completed, the Pt ₁₂ peptide solution is added dropwise while shaking the vial. Then, the solvent is distilled off under reduced pressure with an evaporator as it is in the vial, and vacuum dried at 40 ° C. for 1 day. The obtained Pt ₁₂ metallopeptide-carrying KB was redispersed with MeOH and dropped cast 1 μl × 3 times on a Cu microgrid to prepare a Pt ₁₂ metallopeptide / KB 2 wt% STEM sample.

When the STEM sample was observed with atomic resolution HADDF-STEM, a bright spot of platinum was observed on the KB. A place where 12 bright spots of platinum can be seen spread out as a metallopeptide chain as shown in the frame of FIG. 18, and a place of platinum which is not as small as sub-nanoparticles as surrounded by another frame. It was observed that the two supported states of the place where 12 bright spots are gathered are information from the position of the bright spot of platinum.

In addition, when the expanded and supported area is enlarged, the diameter between platinum and platinum is 5.0 nm, and the diameter between platinum and platinum of the Pt12 peptide considered in the molecular model is about 4.8 nm, which is observed in this sample. From the results of HADDF-STEM, it was confirmed that the bright spots of platinum were due to the Pt ₁₂ peptide (Fig. 19).

STEM observation of Pt ₁₂ sub-nanoparticles
Atomic resolution HADDF-STEM of Pt ₁₂ peptide / KB 2 wt% confirmed that the Pt ₁₂ peptide was supported on the carrier KB. Therefore, a similar sample was placed in an electric firing furnace and placed in a hydrogen atmosphere (H _2). The Pt ₁₂ peptide was reduced under the conditions of / N ₂ 3%), 250 ° C., and 3 hours. Then, the calcined supported KB was placed in a 10 ml vial, redispersed while applying ultrasonic waves to MeOH, and dropped cast on a Cu microgrid 1 μl × 3 times to prepare a STEM sample of Pt ₁₂ sub-nanoparticles / KB 2 wt%. did. When observing the sample, a collection of bright spots that appear to be Pt sub-nanoparticles can be seen in the macro field of view, and when the field of view is expanded with respect to the part surrounded by the yellow frame, Pt sub-nanoparticles with 12 platinum atoms of about 0.9 nm gathered. Since it was seen, the synthesis was successful. By synthesizing Pt ₁₂ sub-nanoparticles this time, the larger the number of metal atoms, the greater the stability as a cluster, and since it is easier to find the sub-nanoparticles on the carrier with HAADF-STEM, many sub-nanoparticles can be observed and the grains. The diameter distribution was 0.91 ± 0.20 nm. When Pt ₁₂ sub-nanoparticles were synthesized using the TPMG4 dendrimer, the value was 0.9 ± 0.1 nm ⁶ , demonstrating that the metallopeptide can be used as a template for sub-nanoparticle synthesis, similar to the TPMG4 dendrimer. .. (Fig. 20)

Synthesis of Pt ₁₉ metallopeptide and Pt ₁₉ sub-nanoparticles
Since Pt ₁₉ sub-nanoparticles have higher ORR activity and catalytic activity than sub-nanoparticles with other atomic numbers, we decided to investigate whether Pt ₁₉ sub-nanoparticles could be synthesized even by using the metallopeptide template method. By newly using NovaNova SynRTGR resin, unlike the case of Trityl OH resin, it is not necessary to extend the first residue by manual synthesis and then perform automatic synthesis, which enables fully automatic synthesis. Initially, a piperidine / DMF 20% (v / v) solution was used as a synthetic pathway to deprotect the terminal Fmoc groups of NovaNovaSynRTGR resin. After that, extension → deprotection was repeated 19 times to synthesize the target sequence NH _2- (E-NH-Pt) ₁₉ -CONH ₂ . (Extension conditions: HATU / DMF 0.2M as a condensing agent, HOBt / DMF 0.2M as a condensing auxiliary agent, N-ethyl-diisopropylamine / NMP 0.2M as an auxiliary base, Fmoc- (E-NH-Pt) -COOH as a condensing peptide / DMF 0.5M, reaction condition: microwave 75 ℃, 5min deprotection condition: piperidine / DMF 20% (v / v) solution) After completion, remove the 5ml vial from the automatic synthesizer and CH ₂ Cl ₂ : TFA: TIPS. = 96.5: 1: 2.5 (v / v) solution was collected performed Kuribeji from the solid phase using cut cocktail, NH ₂ a crude by performing reprecipitation - (E-NH-Pt) 19 - A yellow powder of CONH ₂ was obtained.

Dissolve in CH ₃ CN / H ₂ O 50% (0.1% TFA) solution and analyze from CH ₃ CN / H ₂ O 50% (0.1% TFA) to CH ₃ CN / H ₂ O 95% (0.1%) by Analysis HPLC. When the analysis was performed while applying a gradient to TFA), a large number of peaks were observed (Fig. 21 (a)). As in the case of extending 12 residues, the one with the longest reaction is considered to have the longest retention time, so the main peak in FIG. 21 (a) is NH _2- (E-NH-Pt) ₁₉ -CONH. Assuming the peak of ₂ , fractionation was performed using a preparative column while applying a gradient under the same conditions. The recovered solution was obtained by removing the solvent with a freeze-dryer to obtain a peptide yellow powder of NH _2- (E-NH-Pt) _19- COOH.

When the obtained crude was analyzed by MALDI-TOF-MS, the rising position of the peak was about m / z = 10500, so considering the molecular weight from which PPh ₃ was removed, it was about 15 to 17 residues. It is expected that this is the case (Fig. 21 (b)).

For the separated and lyophilized sample, weigh it with a 182 μg precision balance assuming NH _2- (E-NH-Pt) ₁₉ -CONH ₂ , add it to a 50 ml vial, and then add 10 ml CHCl ₃ to dissolve it. It was. Also, add 10 mg KB to another 50 ml vial, then add 20 ml CHCl ₃ and sonicate for 15 minutes to disperse. After the ultrasonic dispersion is completed, the Pt ₁₉ peptide solution is added dropwise while shaking the vial. Then, the solvent was distilled off under reduced pressure with an evaporator in the vial to prepare Pt ₁₉ metallopeptide / KB 0.47 wt% @ Pt.

Then, the Pt ₁₉ peptide was reduced in an electric firing furnace under a hydrogen atmosphere (H ₂ / N ₂ 3%) at 250 ° C. for 3 hours. The calcined supported KB was placed in a 10 ml vial, redispersed while applying ultrasonic waves to MeOH, and dropped cast 1 μl × 3 times on a Cu microgrid to prepare a STEM sample of Pt ₁₉ SNP / KB 0.47 wt% @ Pt. When observing the sample, fine particles in which platinum atoms were gathered and a single atom of platinum, which seemed to be sub-nanoparticles, were observed. Compared with the case of Pt ₁₂ sub-nanoparticles, many sub-nanoparticles with fewer or more atoms were found (FIGS. 22 (a) and 22 (b)). The reason for this is that the main chain of the peptide becomes longer and the distance between the atoms of Pt-Pt during reduction increases, so that it aggregates with the adjacent sub-nanoparticles during reduction, resulting in a small number of platinum. It is thought that many things and many things have been made.

Evaluation of activity of Pt sub-nanoparticles synthesized by the metallopeptide template method The platinum of the sub-nanoparticle size synthesized in this way was used for the catalytic reaction of the oxidation of toluene shown below.

A catalytic amount (5 mg) of Pt _{19 /} KB0.47 wt% @ Pt supported on 2 ml of toluene was added, and the mixture was reacted in an autoclave at 1 Mpa at 160 ° C. for 5 hours. After the reaction, the reaction product of toluene oxidation was quantified by measuring the solution by NMR. The obtained products were Bendaldehyd, Benzyl Alcohol, Benzoic Acid, and Benzyl Benzoate. When an experiment was conducted using commercially available Pt / C 10 wt%, the yield was 0.23%, but Pt ₁₉ / At KB0.47 wt% @ Pt, the yield was 3.26%, showing 14.2 times the activity (Fig. 23). When converted by TOF, the result was 671 times. From this, it was found that the Pt sub-nanoparticles synthesized by the peptide template method can actually be used as a catalyst.

Synthesis of Fmoc-(E-NH- (PyL-Ir))-OAll Platinum mononuclear metallopeptides have been synthesized, but it is necessary to synthesize metallopeptides having other metal atoms as complexes for alloying. Met. The platinum-carbon bond is very strong and can withstand a strong acid such as TFA, but in the case of a general complex, the acid of TFA may decompose the bond of CM or NM, so the strong acid I thought of a scheme that does not use. At that time, in consideration of developing a metal atom library in the future, it was decided to adopt the pyridine type as the skeleton, which is often reported as a complex. A peptide ligand that does not use a strong acid at the time of complexing is synthesized by condensing phenylpyridine having an amine terminal on the side chain of the glutamic acid derivative.

Add 72 ml of CH ₂ Cl ₂ to Fmoc-Glu-OAl (1.91 g, 4.66 mmol) and EDCl (1.61 g, 8.42 mmol) in an eggplant flask (500 ml) and stir at room temperature for 10 minutes. Then, 4- (pyridine-2-yl) aniline (0.72 g, 4.22 mmol) and DMAP (0.09 g, 0.74 mmol) were added, and the mixture was further stirred at room temperature. After stirring for about 1 hour, a white precipitate began to form. After stirring for 6 hours, MALDI-TOF-MS confirmed the disappearance of the 4- (pyridine-2-yl) aniline peak, so stirring was stopped. The reaction solution was blown off to about one-third with an evaporator and then recrystallized by placing it in a freezer. The white cotton-like material in the solution was filtered through a membrane filter and purified by washing with chilled CH ₂ Cl ₂ . The target product, Fmoc- (E-NH-pyL) -OAl, was obtained in 1.71 g in a yield of 72%. Identification was performed using NMR.
¹ H NMR (CDCl 3, 400 MHz): δ8.60 (d, 1H) δ8.09 (s, 1H) δ7.89 (d, 2H) δ7.69-7.59 (m, 6H)
δ7.53-7.50 (t, 2H) δ7.32 (t, 2H) δ7.22 (t, 2H) δ7.12 (t, 1H) δ5.89-5.77 (m, 1H) δ5.57 (d, 1H) δ5.27 (d. 2H) δ5.20 (d. 2H) δ4.58 (d. 2H) δ4.43-4.33 (m. 3H) δ2.40-2.25 (m. 3H) δ2.20- 1.87 (m.1H)

Since the peptide ligand could be synthesized, the synthesis of Ir metallopeptide was examined using the Ir salt with respect to the side chain phenylpyridine.

Fmoc- (E-NH-pyL) -OAl (81.8 mg, 146 μmol), (Cp ^* IrCl ₂ ) ₂ (38.6 mg, 48.5 μmol) in eggplant flask (100 ml), CH as solvent in sodium acetate (101 mg, 388 μmol) ₂ Add 39 ml of Cl ₂ and stir at room temperature for 1 day. It was confirmed by MALDI-TOF-MS that the peak of Fmoc- (E-NH-pyL) -OAl had disappeared, and stirring was stopped. Then, the undissolved sodium acetate was removed by membrane filter filtration, and the solution was applied to silica gel. After flowing CHCl ₃ as a developing solvent using flash column chromatography, the developing solvent was changed to acetone to obtain a yellow solution. After distilling off the solvent with an evaporator, the target product, Fmoc-(E-NH- (pyL-Ir))-OAl, was obtained in a yield of 30.1% by vacuum drying for 1 day. Identification was performed by NMR and MALDI-TOF-MS (FIGS. 24 (a) and 24 (b)).

Synthesis of Fmoc-(E-NH- (PyL-Ir))-COOH

Fmoc- (E-NH-Pt) -OAll (1.09 g, 1.18 mmol) and Pd (PPh ₃ ) (0.12 mg, 0.10 mmol) synthesized in a two-port eggplant flask (100 ml) in the same manner as above, dimedone ( 0.34 mg, 2.42 mmol) is added to replace Ar in the system. A septum rubber was attached to one side, and a three-way cock was attached to the other side. Dehydrated CH ₂ Cl ₂ was added with a 20 ml syringe, and the inside of the system was replaced with Ar again. A balloon was attached to the tip of the three-way cock, and the mixture was stirred at room temperature for 3 hours while being kept under Ar. After directly charging the upper part of the silica gel column by flash column chromatography, 10 CV was flowed with CHCl ₃ , and then the developing solvent was flowed while applying a gradient at 10 CV until CHCl ₃ : MeOH = 90: 10 to separate the target product. did. The solvent of the separated fraction was distilled off under reduced pressure and dried under vacuum for 1 day to obtain a yellow powder. When the purity was measured by analytical HPLC, it was 77% (Fig. 25).

Solid-phase attachment / detachment test of Fmoc-(E-NH- (PyL-Ir))-COOH It was investigated whether the synthesized Fmoc-(E-NH- (PyL-Ir))-COOH can be applied under weak acid conditions.

First, a piperidine / DMF 20% (v / v) solution was used to deprotect the terminal Fmoc groups of NovaNovaSynRTGR resin. Thereafter, elongation → deprotection performs _{NH 2 - (E-NH- (} pyL-Ir)) - were synthesized sequence of CONH _2. For elongation, HATU / DMF 0.2M as a condensing agent, HOAt / DMF 0.2M as a condensation aid, N-ethyl-diisopropylamine / NMP 0.2M as an auxiliary base, and Fmoc- (E-NH-Pt) -COOH / as a condensing peptide. DMF 0.03M was added, and the conditions were microwave 75 ° C. and 5 min. The deprotection of the terminal Fmoc group was reacted with a piperidine / DMF 20% (v / v) solution. After completion, remove the 5 ml vial from the automatic synthesizer, cleave from the solid phase using a cut-out cocktail of CH ₂ Cl ₂ : TFA: TIPS = 96.5: 1: 2.5 (v / v), collect the solution, and re-collect. a crude by performing precipitation operation _{NH 2 - (E-NH- (} pyL-Ir)) - to give a yellow powder CONH _2.

Dissolve in CH ₃ CN / H ₂ O 30% (0.1% TFA) solution and analyze from CH ₃ CN / H ₂ O 30% (0.1% TFA) to CH ₃ CN / H ₂ O 95% (0.1%) by Analysis HPLC. When the analysis was performed while applying a gradient to TFA), peaks were observed at all positions similar to those of the raw material, indicating that Ir metallopeptide can tolerate excision under weak acid conditions (Fig. 26).

Next, the synthesis of alloy metallopeptides and sub-nanoparticle formation were investigated.
Synthesis of Pt ₃ Ir ₃ Metallopeptides Initially, a piperidine / DMF 20% (v / v) solution was used to deprotect the terminal Fmoc groups of Nova Nova SynRTGR resin. After that, the extension of Fmoc- (E-NH-Pt) -COOH → deprotection was repeated 3 times, and then the extension of Fmoc-(E-NH- (pyL-Ir))-COOH → deprotection was repeated 3 times. The sequence of NH _2- (E-NH-Pt) _3- (E-NH-(pyL-Ir)) ₃ -CONH ₂ was synthesized (extension condition: HATU / DMF 0.2M as a condensing agent, HOBt / DMF 0.2M as a condensation aid, N-ethyl-diisopropylamine / NMP 0.2M as an protecting group, Fmoc- (E-NH-Pt) -COOH / DMF 0.04M as a condensing peptide, Fmoc- (E-NH-(E-NH- pyL-Ir))-COOH / DMF 0.04M, reaction condition: microwave 75 ℃, 5min deprotection condition: piperidine / DMF 20% (v / v) solution). After completion, remove the 5 ml vial from the automatic synthesizer, cleave from the solid phase using a cut-out cocktail of CH ₂ Cl ₂ : TFA: TIPS = 96.5: 1: 2.5 (v / v), collect the solution, and re-collect. By performing the precipitation operation, a yellow-orange powder of NH _2- (E-NH-Pt) _3- (E-NH-(pyL-Ir)) ₃ -CONH ₂ was obtained. Dissolve in CH ₃ CN / H ₂ O 30% (0.1% TFA) solution and analyze from CH ₃ CN / H ₂ O 30% (0.1% TFA) to CH ₃ CN / H ₂ O 95% (0.1%) by Analysis HPLC. When the analysis was performed while applying a gradient to TFA), many peaks were observed (Fig. 27). Therefore, the HPLC preparative (7 ml / min) was divided into 1 ml increments to collect fractions. When each fraction was analyzed by MALDI-TOF-MS, the peak of the target substance could be observed from the fraction at the position of Retention time = 27.4, so the solution was freeze-dried and recovered.

Synthesis and reduction of Pt ₃ Ir ₃ sub-nanoparticles Electronic state before and after synthesis Synthesized NH _2- (E-NH-Pt) _3- (E-NH- (pyL-Ir)) _3- CONH ₂ (52 μg, 12 nmol) Prepare a 230 μM Pt ₃ Ir ₃ peptide solution by dissolving in 52 μl of chloroform, drop cast the solution 1 μL × 3 times onto the HOPG substrate, measure XPS, and place the measured substrate in an electric tube furnace. Baking was performed at 250 ° C while flowing gas under the condition of H ₂ / N ₂ 3%.

Comparing the XPS before and after firing, the binding energies of Pt 4f _7/2 peak and Pt 4f _5/2 peak were 73.0eV and 76.3eV, respectively, before firing, but after firing, Pt 4f _{7 /} The binding energies of the ₂ peaks and the Pt 4f _5/2 peak became 71.3eV and 74.5eV, and the binding energy shifted by about 1.5eV as in the case of firing the Pt ₄ peptide. Therefore, by firing in a hydrogen atmosphere, Pt was increased. It was shown that it can be reduced from a divalent state to a zero-valent state. In addition, the binding energies of Ir 4f _7/2 peak and Ir 4f _5/2 peak were 63.4eV and 66.7eV before firing, respectively, but after firing, the binding energies of Ir 4f _7/2 peak and Ir 4f _5/2 peak were Since the binding energies became 62.3eV and 65.1eV, and the binding energy shifted by about 1.3eV like platinum, it was shown that Ir can be reduced from a trivalent state to a zero-valent state by firing in a hydrogen atmosphere. (Fig. 28). The measured value is charge-up corrected based on 284.5 eV of C 1s based on HOPG.

TEM observation and EDS spectrum measurement of Pt ₃ Ir ₃ sub-nanoparticles
NH _2- (E-NH-Pt) _3- (E-NH-(pyL-Ir)) ₃ -CONH ₂ Weigh 20 μg with a precision balance and add to a 10 ml vial, then add 2 ml of CHCl ₃ to dissolve. It was. Also, add 1 mg KB to another 10 ml vial, then add 2 ml CHCl ₃ and sonicate for 15 minutes to disperse. After the ultrasonic dispersion is completed, the Pt ₃ Ir ₃ peptide solution is added dropwise while shaking the vial. Then, the solvent was distilled off under reduced pressure with an evaporator in the vial to prepare Pt ₃ Ir metallopeptide / KB 2 wt%.

Then, the mixture was placed in an electric firing furnace and reduced to Pt ₃ Ir ₃ metallopeptide under a hydrogen atmosphere (H ₂ / N ₂ 3%) at 250 ° C. for 3 hours. The calcined supported KB was placed in a 10 ml vial, redispersed in MeOH while ultrasonically applied, and dropped cast 1 μl × 3 times onto a Cu microgrid to prepare a STEM sample of Pt ₃ Ir ₃ sub-nanoparticles / KB 2 wt%. When observing the sample with HAADF-STEM, bright spots that are thought to be sub-nanoparticles are seen on the KB, and when enlarged, 6 bright spots that are thought to be Pt ₃ Ir ₃ sub-nanoparticles are observed at a size of about 0.6 nm. It succeeded (Fig. 29 (a), (b)).

In addition, when EDS mapping was performed in another field of view, the elements of Ir and Pt could be obtained as mapping from one sub-nanoparticle, and in the EDS spectrum, Cu, Pt, and Ir derived from the STEM sample substrate were partially spectrumed. Although they overlapped, two kinds of elements were separated separately by the Ir La and Pt La lines (FIGS. 30 (a) and 30 (b)).

From the XPS results, the elements of Pt and Ir exist before and after the reduction, and the binding energy shifts by about 1.5 eV before and after the reduction. Therefore, both Pt and Ir were reduced from the electronic state of the complex to the metal. It was confirmed. In addition, from the STEM results, we confirmed a collection of 6 bright spots that are thought to be Pt ₃ Ir ₃ sub-nanoparticles on the KB, and obtained the characteristic X-ray spectra of Pt and Ir from EDS. Therefore, we used the peptide template method. We have succeeded in synthesizing alloy sub-nanoparticles.

Claims (4)

A metallized peptide containing the plurality of metal atoms in which the same or different metal atoms are bonded to a plurality of the same or different amino acid residues is reduced or hydrolyzed, or chemically converted accordingly. A method for producing a metal-containing cluster, which forms a metal-containing cluster composed of the plurality of metal atoms and / or a metal oxide derived from the metal atom. The metal-containing cluster according to claim 1, wherein the metallized peptide is supported on the surface of the carrier and then reduced or hydrolyzed, or chemically converted according to these to form the metal-containing cluster on the surface of the carrier. Method for producing metal-containing clusters. The metal according to claim 1 or 2, wherein the structure in which a metal atom is bonded to the amino acid residue is a structure in which the metal salt compound (A) and the amino acid residue (B) are bonded by an amidation reaction. Method for producing contained clusters. The method for producing a metal-containing cluster according to any one of claims 1 to 3, wherein the metallized peptide is synthesized by a solid-phase synthesis method.