WO2007097406A1 - Hydrophilic carbon cluster - Google Patents

Abstract

Disclosed is a method for converting a carbon cluster into a hydrophilic carbon cluster without greatly changing the characteristics of the carbon cluster. Also disclosed are a hydrophilic carbon cluster having a structure wherein a substituted or unsubstituted amino group is bonded to a carbon atom of a carbon cluster, an aqueous dispersion of such a hydrophilic carbon cluster, and a solid phase synthesis using such a hydrophilic carbon cluster.

Description

 Hydrophilic carbon cluster

 The present invention relates to a hydrophilic carbon cluster, a carbon cluster dispersion, a method for producing a hydrophilic carbon cluster, and a solid-phase synthesis method using the hydrophilic carbon cluster. Background Technology-Carbon clusters with nanometer-scale microstructures such as carbon nanotubes and carbon nano-light horn opifullerenes are expected to be applied to new electronic materials, catalysts, optical materials, nanotechnology, etc. Nanostructured graphite (graphite) material (S. Iijima, et al., Nature, 1991, 354, 56, S. Iijima et al., Nature, 1993, 363, 603, S. Iijima et al , 1999, Chera. Phys. Lett., 1999, 309, 165). For example, carbon nanotubes can be either metals or semiconductors, depending on their structure, and research is underway to create nanometer-sized devices.

 However, the lack of dispersibility of carbon clusters in water is a problem for application to medical applications. Therefore, a method for dispersing in an aqueous solvent is being studied. Recently, methods have been developed to prepare aqueous dispersions by complexing with noncovalent bonds using solubilizing agents such as surfactants and water-soluble polymers (O 'Connell, MJ et al., Science 2002, 297, 593, Zheng, M. et al., Nature Mat. 2003, 2, 338). However, these solubilizers are easily dissociated from carbon clusters in aqueous solution, and many are toxic to living organisms. Japanese Patent Application Laid-Open No. 2 0 0 5-1 0 4 7 6 2 discloses that carbon nanotubes are stably solubilized in an aqueous solvent by complexing them with a polysaccharide j3_1,3-glucan. A method is disclosed, and Japanese Patent Application Laid-Open No. 2 085-285 60 discloses a method of solubilizing carbon nanotubes using DNA and oligonucleotides. However, since these methods require a large amount of solubilizing agent, it is thought that the characteristics of carbon clusters will be greatly modified. Furthermore, Japanese Patent Application Laid-Open No. 2003-009 5 6 2 4 discloses a method for preparing a hydrophilic carbon nanohorn by introducing an oxygen functional group such as a carbonyl group and further chemically modifying it. It is disclosed. However, this method oxidizes carbon nanohorn, and this method cannot introduce an amino group.

The solid-phase synthesis method is a method in which an organic compound is introduced onto the surface of a solid and then chemically modified. This is a technique that enables the target compound to be isolated by separating the reaction reagent / solvent by filtration of the solid phase carrier and then cutting it out from the carrier. Since it can be easily purified and isolated, it is used in the synthesis of various organic compounds. At present, polystyrene resin and glass particles As a solid support, it has been used in the synthesis of peptide DNA and in recent years in combinatorial chemistry for the synthesis of various compounds.

 The solid phase carrier widely used in the solid phase synthesis is usually micrometer-sized resin particles (beads), and the reaction is carried out by gently shaking. In addition to the reaction on the solid surface, the reaction efficiency may be low because it is physically weak and cannot be vigorously stirred. In addition, in order to analyze the reaction product obtained by the solid phase synthesis method, it is necessary to cut out from the support, and it is complicated to confirm the success or failure of the reaction at each reaction stage. For this reason, recently, the development of a method for direct analysis of compounds on a solid support has attracted attention. Disclosure of the invention

 An object of the present invention is to provide a method for converting a carbon cluster into a hydrophilic carbon cluster without greatly modifying the properties of the carbon cluster.

 As a result of intensive studies to solve the above problems, the present inventors have found that a hydrophilic carbon cluster that can be dispersed in an aqueous solvent can be produced by allowing a metalamide to act on the carbon cluster. It came to complete. ,

 That is, the present invention includes the following inventions.

 (1) A hydrophilic carbon cluster having a structure in which a substituted or unsubstituted amino group is bonded to a carbon atom of the carbon cluster.

 (2) The hydrophilic carbon cluster according to (1), wherein the amino group is an unsubstituted amino group.

 (3) The hydrophilic carbon cluster according to (1) or (2), wherein the carbon cluster is a tubular carbon cluster.

 (4) The hydrophilic carbon cluster according to (3), wherein the carbon cluster is carbon nanohorn, and a substituted or unsubstituted amino group is bonded to a carbon atom of at least a conical end cap.

(5) A carbon cluster dispersion in which the hydrophilic carbon cluster according to any one of (1) to (4) is dispersed in an aqueous solvent.

 (6) A method for producing a hydrophilic carbon cluster having a structure in which a substituted or unsubstituted amino group is bonded to a carbon atom of the carbon cluster, which comprises reacting the carbon cluster with a metal amide.

(7) The method according to (6), wherein the carbon cluster is a tubular carbon cluster.

 (8) The method according to (6) or (7), wherein the metal amide is an alkali metal amide.

 (9) The method according to any one of (6) to (8), further comprising chemically modifying an amino group bonded to a carbon atom of the carbon cluster.

 (10) A carrier for use in solid phase synthesis of an organic compound comprising the hydrophilic carbon cluster according to any one of (1) to (4).

 (11) A carrier for use in solid phase synthesis of organic compounds,

(1) to the amino group of the hydrophilic carbon cluster according to any one of (4) to the solid phase synthesis ligand The carrier having a structure in which a carrier molecule is covalently bonded.

 (1 2) A method for solid-phase synthesis of an organic compound using the carrier according to (1 0) or (1 1).

(1 3) The method according to (1 2), wherein the organic compound is a polypeptide or a polynucleotide. According to the present invention, a carbon cluster can be converted into a hydrophilic carbon cluster without greatly modifying the properties of the carbon cluster. In addition, by using the obtained hydrophilic carbon cluster for solid-phase synthesis, synthesis reaction and product analysis can be performed under the same conditions as solution reaction.

 This specification includes the contents described in the claims and the drawings of Japanese Patent Application No. 2006-042 872, which is the basis of the priority of the present application. Brief description of the drawings.

Figure 1 shows the process of producing an amino group NHA 2 having an amino group in the end cap of a cone from carbon nanohorn aggregates (NHA). The amino group of aminated NHA 2 is converted to 1-fluoro-2, 4- An outline of a step of producing aminated NHA 3 by converting it to a dinitrophenol group with dinitrile benzene, and a step of producing a fluorescently labeled aminated NHA 4 from the aminated NHA 2 are shown. Figure 2 shows the IR spectra of untreated NHA (top) and aminated NHA2 (bottom). A broad signal around 3200 cm- ¹ indicates the presence of an amino group in the aminated NHA2.

 Figure 3 (A) shows a TEM image of aminated NHA2. It can be seen that the conical shape of the carbon nanohorn is maintained. The scale par represents 2 nm. (B) shows a photograph of an aqueous solution of aminated NHA 2 at concentrations of 1.0 mg / mL and 0.20 mg / mL and untreated NHA (from left to right). (C) shows DLS data of an aqueous solution of aminated NHA 2 (0.20 mg / mL). (D) shows an AFM image of Aminig NHA 2 deposited on My force. A scale par represents 500 nra.

 Figure 4 shows the results of size analysis of aminated NHA 2 by AFM.

 Figure 5 (A) shows the UV-vis spectrum of aminated NHA 2 at a concentration of 0.02 mg / mL. The absorption at 260 nm corresponds to the plasmon absorption of the NHA graphite structure. (B) shows the UV-vis spectrum of aminated NHA 3 at a concentration of 0.02 mg / raL. The inset shows the differential spectrum of aminated NHA 3 and aminated NHA 2. The absorption at 350 nm corresponds to a dinitrophenylamino group, demonstrating the presence of an amino group on NHA. (C) shows the 1 ^ 115 spectrum of the amination 11 ^ 4 at a concentration of 0.021 ^ / 111. The inset shows the differential spectra of aminated NHA 4 and aminated NHA 2. Absorption at 500 nm corresponds to Oregon Green 488.

Fig. 6 (A) shows a chromatogram of aminated NHA 2 (0.2 rag / raL, 0.5 mL) eluted with Cefacryl 500HR (resin volume: 17 raL, detection: 260 nra). The positions of the fractions are shown in the order of fractions 22: red, 23: orange, 24: green, 27: blue. (B) shows typical data of DLS analysis of fractions. The color representing the fraction is the same as A, and in order from the top, it is represented by the colors of fraction 22: red, 23: orange, 24: green, and 27: blue. FIG. 7 shows a photograph of the column after elution of the aminated NHA 2. (A) shows a photo of the column when Cefacryl 100HR is used, (B) shows when Cefacryl 500HR is used, and (C) shows a column photo when Cefacryl 1000SF is used. Aminated NHA 2 was dissolved from the Cefacryl 500HR column but did not elute from the other columns.

 Figure 8 shows the excitation (red) and emission (blue) spectra of aminated NHA 4 indicating the presence of a fluorescent dye.

 Figure 9 (A) shows the survival rate when 3T3 cells and HeLa cells were cultured with particles. This survival rate was calculated using the Bradford method. (B) shows a photograph of 3T3 cells cultured. The photo on the left does not contain aminated NHA 2, and the photo on the center and right show the addition of aminated NHA 2 at concentrations of 0.1 rag / mL and 1 mg / mL, respectively.

FIG. 10 shows the ¹ H MR spectrum of N-Fmoc-Gly-HMP-AN synthesized in Example 11. FIG. 11 shows the 匪 F spectrum of N-Fmoc-Ala-ΗΜΡ-ΑΝ synthesized in Example 12. BEST MODE FOR CARRYING OUT THE INVENTION

 In the present invention, the carbon cluster refers to an aggregate formed by bonding or aggregation of several to several hundred carbon atoms, regardless of the type of carbon-carbon bond. However, it is not necessarily limited to those composed only of 100% carbon, but also includes those mixed with other atoms. Including such a case, an aggregate in which a large number of carbon atoms occupy is referred to as a carbon cluster. Carbon clusters include spherical carbon clusters, such as fullerenes, and tube-like carbon clusters, such as carbon nanotubes, striking bon nanohorns, and carbon fiber derivatives, and their chemically modified derivatives and their Aggregates are included. A carbon nanotube, which is one of the tube-like carbon clusters, is a multiple tube made by stacking 2 to several tens of layers of graphitic carbon, and each layer has a closed structure like fullerene. As carbon nanotubes, single-walled carbon nanotubes (SWCNT) consisting of single-walled tubes and multi-walled carbon nanotubes (MWCNTs) in which two or more layers are concentrically overlapped are known. Can be used. The single-bonn nanotube and its derivatives can be obtained by known production methods such as arc discharge (Ac Chem. Res. Vol. 16, no. 12, 2002, p. 1035-1044), heat Decomposition method (J. Phys. Chera. B vol. 105, no. 35, 2001, p. 8297-8301), laser evaporation 'method (Carbon vol. 33, no. 7, 1995, p · 903-914), It can be obtained by a gas phase synthesis method (Science vol. 306, no. 5700, 2004, p. 1362-1364). The average fiber length of carbon nanotubes (which can be determined from the average number of major axis lengths obtained from image analysis of electron micrographs) is preferably 10 to 100 μιη, and the diameter is 0.8 to 20 It is preferable that it is nm. .

Tsukichi Bonn Nanohorn has a hollow conical shape, that is, a horn-like structure in which the tube diameter is continuously increased and the tube diameter is continuously increased, unlike carbon nanotubes. No. 2001-64004). Carbon nanohorns can be produced by the same method as carbon nanotubes. For example, as disclosed in the above patent gazette, a structure in which spherical particles are gathered by irradiating a solid carbon simple substance with a laser beam in an inert gas atmosphere and evaporating the carbon laser. Can be obtained as These spherical particles are aggregates of single-walled carbon nanotubes whose tip is closed in a conical shape. Some of them have shapes resembling flowers of dahlia, buds, and ridges.

As one embodiment, a preferred method for producing carbon nanohorn will be described below. A solid carbon simple substance is irradiated with laser light in an inert gas atmosphere to evaporate carbon laser to obtain a powder in which spherical substances are aggregated as soot-like substance. Carbon laser evaporation, Ar (Al gon), the He in a reaction inert gas atmosphere including a rare gas (Heriumu), etc., the surface of the solid carbon single substance laser beam such as a high output C0 ₂ gas laser beam Irradiate at an appropriate angle. As the carbon simple substance as a solid substance, for example, round bar-like sintered carbon, compression molded carbon, or the like can be used. The soot-like substance can be collected by being deposited on an appropriate substrate or collected by a dust bag. This soot-like substance can be recovered by flowing an inert gas in the reaction vessel and flowing the inert gas. The obtained soot-like substance is a particle in which carbon nanohorn structures are aggregated to form spherical particles, and a large number of these spherical particles are aggregated. This can be a single or a collection of spherical particles. In this case, the spherical particles in a single or a plurality of aggregated states are recovered by suspending in, for example, ethanol as a solvent, performing ultrasonic stirring and decantation, and repeating as necessary. it can.

 Commercially available tube-like carbon clusters such as carbon nanotubes and carbon nanohorns may be used. For example, Honjo Chemical Co., Ltd., Tokyo Progress System Co., Ltd., Science Laboratories Co., Ltd., Carbon Co., Ltd. · Nanotech · Research · Institute (CNRI), US · Hyperion and other mixed products and refined products.

 Fullerenes are spherical shell-like carbon molecules that contain a 5-membered ring in addition to the 6-membered ring found in graphite in the network of carbon atoms. For example, C36, C60, C70, C76, C78, C80, C82, C84, C86, C84, C88, C90, C92, C94, C96, and the number of carbon atoms in one molecule exceeds 96 and the maximum aggregate mass is 30 Higher order fullerenes of nm or less, and mixtures of two or more of these. Of these, C60, C70, C76, and C82 are preferably used. These fullerenes can be synthesized by a known method.

For example, a method for producing C36 is described in New Daiamond. Vol. 16, no. 2, 2000, p. As a method for producing C60, C70, C76, C78, C82, C84, C90 and C%, J. Phy. Chem., 94, 8634 (1990) describes a production method by an arc discharge method, and Z. Phys. D, 40, 414 (1997) describes the production method by the oven laser method. Higher order fullerenes with more than 96 carbon atoms in one molecule and a maximum aggregate size of 30 nra or less are It can be obtained as a byproduct of the coke discharge method.

Commercially available fullerenes include those made by Frontier Carbon Co., Ltd. and MATERIALS TECHNOLOGIES RESEARCH MTR LIMITED for C60 and C70, and those made by MATERIALS TECHNOLOGIES RESEARCH MTR LIMITED for C76, C78 and C8. It is done. In the present invention, a mixture of fullerenes having different carbon numbers may be used. Commercially available products include Frontier Carbon Co., Ltd., Science Laboratories Co., Ltd., Tokyo Progress System Co., Ltd., MATERIALS TECHNOLOGIES RESEARCH MTR LIMITED, Inc. Of C60 / C70. Carbon cluster derivatives include those obtained by chemically modifying carbon clusters with functional groups. Examples of the functional group to chemically modify the carbon cluster, for example, alkyl, C ₂ - ₆ § alkenyl, C ₂ - ₆ alkynyl, carboxyl, human Dorokishiru, epoxy, etc. talent Kisentan the like. Carbon cluster derivatives include those obtained by chemically modifying carbon clusters with metal elements. Known metal elements that chemically modify carbon clusters can be used, for example, alkali metals such as Li, Na and K, alkaline earth metals such as Be, Mg and Ca, Ti, V, Examples include transition metals such as Cr, Mn, Fe, Co, Ni, Cu and Zn. ,.

The hydrophilic carbon cluster of the present invention has a structure in which a substituted or unsubstituted amino group is bonded to the carbon atom constituting the carbon cluster. Hereinafter, the hydrophilic carbon cluster of the present invention may be referred to as an aminated carbon cluster. Substituted or unsubstituted amino groups - are tables in NR ^2, wherein, R ¹ and R ² are each independently a hydrogen atom, a halogen, hydroxyl, - ₆ alkyl, C ₂ - ₆ alkenyl, C ₂ - Selected from ₆ alkynyl, d- ₆ alkoxy, _ ₆ acyl, aryl, heteroaryl and cycloalkyl. The alkyl, c ₂ - ₆ alkenyl, c ₂ - ₆ alkynyl, _d-6 alkoxy, c, _ ₆ Ashiru, Ariru, Heteroariru and cycloalkyl le may be substituted.

In this specification, _<6 alkyl, C ₂ _ ₆ alkenyl, C ₂ _ ₆ alkynyl, ₍₆ alkoxy Oyo Pi - ₆ Ashiru an alkyl group, an alkenyl group containing carbon atoms of the number of described respectively, alkynyl Group, alkoxy group and acyl group, which may be linear or branched.

 As used herein, aryl represents an aromatic monocyclic or polycyclic carbon containing 5 to 20 carbon atoms, preferably 6 to 14 carbon atoms, more preferably 6 to 10 carbon atoms. A hydrogen ring group is meant. Examples of aryls include, but are not limited to, phenyl, naphthyl, indur, azleninole, fluorenyl, anthracenyl, phenanthrenol, tetrahydronaphthyl, indanyl and phenanthridinyl.

In the present specification, heteroaryl means an aromatic monocyclic ring group or a polycyclic ring group, wherein the aromatic monocyclic ring group or polycyclic ring group is 5 to 2 h. Carbon atoms, preferably 5 to 10 carbon atoms, wherein one or more ring carbons, preferably 1 to 4 ring carbons, respectively, oxygen atom, nitrogen atom or sulfur atom, etc. Has been replaced by a heteroatom. Preferred heteroaryls include 5-6 membered monocyclic heteroaryl and 8-10 membered bicyclic heteroaryl. Examples of heteroaryl include, but are not limited to, imidazolyl, quinolyl, isoquinolyl, indolyl, indazolyl, pyridazyl, pyridyl, pyrrolyl, pyrazolyl, pyrazinyl, quinoxalyl, pyrimidinyl, pyridazinyl, furyl, chinyl, triazolyl, azolyl, azolyl, Ruporinyl, tetrazolyl, benzofuranyl, oxazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, furazanyl, oxadiazolyl, benzoimidazolyl, benzocenyl, quinolinyl, benzotriazolyl, benzothiazolyl, Examples include isoxazolyl.

 Cycloalkyl as used herein is a monocyclic or polycyclic non-aromatic having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms. This means a hydrocarbon ring group.

Relates R ¹ and R ^2, above ₍₆ alkyl, C ₂ _ ₆ alkenyl, C ₂ - ₆ alkynyl, - ₆ an alkoxy, ₆ Ashiru, Ariru, as the substituents of Heteroariru Contact Yopi cycloalkyl, halogen, hydroxy Honoré , nitro, amino ,, mercapto, Shiano, Isoshiana one preparative, carboxyl, - ₆ alkyl, C ₂ - ₆ alkenyl, _d-6 alkoxy, cycloalkyl, cycloalkylalkyl, Ariru, Heteroariru, C _1-6 alkylamino, C _1-6 alkylthio and the like.

In the aminated carbon cluster of the present invention, preferably either R ¹ or R ² is a hydrogen atom, more preferably both are hydrogen atoms.

 A structure in which a substituted or unsubstituted amino group is bonded to a carbon atom of a carbon cluster means that a substituted or unsubstituted amino group is covalently bonded to a carbon atom constituting the carbon cluster.

 A substituted or unsubstituted amino group is considered to be preferentially bonded to a highly reactive site in the carbon cluster. When there is a local bond structure that is different from the 6-membered ring in the graphite, such as a 5-membered ring or 7-membered ring, a finite curvature is created, which not only changes the geometrical shape of the material, It is thought that a new electronic structure will be generated and the reactivity and reaction rate will increase.

The surface of carbon nanotubes is usually covered with a six-membered graphite structure, but if the five-membered or seven-membered ring is mixed with this six-membered ring, the diameter of the tube will be reduced or expanded. It has been known. Conical carbon nanohorns have a horn diameter that changes continuously, making the surface graphite structure more irregular than carbon nanotubes and adding amino groups. Since the carbon cap of the carbon nanohorn is also highly reactive, when the carbon cluster is a carbon nanohorn, the substituted or unsubstituted amino group is at least a carbon atom present in the end cap of the carbon nanohorn cone. It is thought that they are combined. The amount of substituted or unsubstituted amino groups contained in the hydrophilic carbon cluster of the present invention is determined by the method of Sanger (Paquette, LA Eds. Wiley, Encyclopedia of Reagents Organic Synthesis Vol. 4, pp 2556-2557, 1995). When measured, it is usually 0.1 to 0.3 μmol / rag, preferably 0.21 to 0.27 ^ raol / rag. This corresponds to a density of one amino group in the surface area of the carbon cluster of usually 100 to 500 nm ² , preferably 200 to 400 nm ² .

 The present inventors have also found that substituted or unsubstituted amino groups contained in hydrophilic carbon clusters react with ninhydrin. Therefore, the substituted or unsubstituted amino group contained in the hydrophilic carbon cluster can be obtained using the ninhydrin color reaction (Madeleine, MJ; Tracy, RT; Norman, HN Tetrahedron. 1991, 47, 8791-8830). It can be easily quantified. Similar to the reaction with ordinary amino acids, imine is formed from the reaction of the amino group and ninhydrin at the initial stage of the reaction. On the carbon skeleton of the aminated carbon cluster, it is thought that in addition to the amino group, there is a hydrogen atom or a negative charge generated in equilibrium from it. For this reason, from a carbon cluster with an imine moiety, the elimination of the imine moiety can proceed by a reaction from a negative charge on the carbon skeleton. The 2-imino-1,3-indazione produced by this reaction reacts with another molecule of ninhydrin to produce ruheraann purple. ,

 Specifically, aminated carbon clusters are mixed with ninhydrin hydrate and phenol opium cyanide in an ethanol / piperidine mixed solvent, and after the reaction, the compounds derived from the carbon clusters are added to membrane filter paper (fine filter paper). Removal with a pore size of 0.2 μπι) gives a purple solution. The amino group can be quantified from the absorption value at 570 nm in the UV-visible spectrum of this solution.

 In the present invention, the hydrophilic property means that the aminated carbon cluster of the present invention can be dispersed or dissolved in an aqueous solvent. The hydrophilic carbon cluster 1 of the present invention can be dispersed in water at a concentration of 1 mg / mL or more.

 The hydrophilic carbon cluster of the present invention, except for the property of being hydrophilic, maintains the properties of the carbon cluster before introducing a substituted or unsubstituted amino group, such as a three-dimensional structure, an aggregated structure, and a function. Yes.

By dispersing the hydrophilic carbon cluster of the present invention in an aqueous solvent, a carbon cluster dispersion can be obtained. By performing ultrasonic treatment, it can be dispersed more efficiently. The carbon cluster dispersion is not particularly limited as long as carbon clusters are uniformly dispersed in an aqueous solvent, and includes an aqueous solution of carbon clusters. The carbon cluster dispersion of the present invention usually contains carbon clusters at a concentration of 0.001 to 2 mg / mL, preferably at a concentration of 0.001 to 1 rag / mL. The aqueous solvent is not limited as long as it is a water-based solvent, and water is usually at least 50% by weight, preferably 70% by weight. /. Contains above. The aqueous solvent may contain alcohol, dimethyl sulfoxide (DMS0), tetrahydrofuran (THF), dimethylformamide (DMF) and the like in addition to water. The carbon cluster dispersion of the present invention usually contains particles having an average particle size of 10 to 5000 nm, preferably 100 to 300 nm, more preferably 120 to 250 nm, as measured by dynamic light scattering (DLS). . The particles preferably have a spherical shape. The carbon cluster dispersion of the present invention is advantageous in that the particle size distribution is small. Therefore, it can be suitably used as standard particles. The range of the particle size distribution is usually 50 to 500 nra, preferably 90 to 160 nm.

 This effort also relates to a method for producing hydrophilic carbon clusters. The present inventors have found that a hydrophilic carbon cluster or star in which a substituted or unsubstituted amino group is bonded to a carbon atom can be produced by reacting a metal amide with a carbon cluster. Carbon clusters and metal amide are usually mixed and reacted in a solvent. .

The metal amide can be considered as a salt of NH-ion, and preferably an alkali metal amide and an alkaline earth metal amide are used. The metal amide is represented by MNR ² or M (NR ² ) ₂ . Here, R ¹ and R ² are as described above, preferably a hydrogen atom. M represents a metal, preferably an alkali metal (eg, Li, Na or K) or an alkaline earth metal (eg, Be, 'Mg or Ca). Preferred examples of the alkali metal amide include sodium amide (NaNH ₂ ), potassium amide (KN¾), lithium amide (LiNH ₂ ), and lithium jetyl amide ((C ₂ H ₅ ) ₂ NLi). . Preferred alkaline earth metal amides include magnesium amide (Mg (N¾) ₂ ) and calcium amide (Ca (NH ₂ )). Preferably, sodium amide is used.

 The reaction solvent is not particularly limited as long as it can disperse carbon clusters. For example, liquid ammonia, black benzene, dimethylformamide, dimethylsulfoxide and the like can be used. Liquid ammonia is preferably used as a solvent because it has a low boiling point and can be easily removed after the reaction. The reaction temperature is not particularly limited as long as it is a temperature not higher than the boiling point of the solvent. The force is usually -78 to 180 ° C. The reaction time is usually 1 to 6 hours, preferably 3 to 4 hours. In the case of mass synthesis, it is usually 3 to 9 hours, more preferably 5 to 7 hours. The mixing ratio of the carbon cluster and the metal amide is not particularly limited. The metal amide is usually added in an amount of 1 to 5 g, more preferably 1 to 3 g with respect to the force S and the carbon cluster lg. In the case of mass synthesis, 2 to 6 g, more preferably 2 to 5 g of metal amide is added to 1 g of carbon cluster. The resulting crude product can be purified by methods commonly used in the art.

 The aminated carbon cluster obtained by the above method can further chemically modify the bonded amino group. Examples of such chemical modification of an amino group include amidation, alkylation, arylation, azination, and diazotization.

The aminated carbon cluster of the present invention can be used as a solid support in solid phase synthesis of organic compounds. The carrier for solid phase synthesis of the present invention preferably has a structure in which a single molecule of a linker for solid phase synthesis is covalently bonded to the amino group of the aminated carbon cluster of the present invention. The carrier for solid phase synthesis of the present invention is used in the synthesis of polypeptides and polynucleotides, etc. For example, it can be used as a starting point in solid-phase synthesis in which structural units of organic compounds to be synthesized are continuously added.

 The carrier for solid phase synthesis of the present invention is suitably used for solid phase synthesis of polypeptides and polynucleotides as organic compounds. It is also preferably used in combinatorial chemistry. In solid-phase synthesis, organic compounds to be synthesized, for example, structural units of polypeptides and polynucleotides, such as amino acids, nucleosides, and derivatives thereof, are linked sequentially.

 In the present specification, the polypeptide refers to a compound formed by forming a peptide bond with amino acid, and includes oligopeptides. The amino acid is not particularly limited as long as it is an organic compound having both an amino group and a carboxyl group, but is preferably an α-amino acid. Polynucleotides also include oligonucleotides, including nucleic acids (DNA and RNA) and derivatives thereof. Nucleic acid derivatives include those known in the art, and phosphorothioate type, phosphorodithioate type, phosphorodiamidate type, methylphosphonate type, methylphosphonate in which the oxygen atom of the phosphate moiety is replaced with a sulfur atom. Notioate type, Substituent modified type on furanose ring, Pyranose type with 1-carbon increase in sugar ring skeleton, Polycyclic sugar skeleton type, Pyrimidine C-5 position modified base type, Purine C-7 position modified base type And nucleic acids with extended ring-modified bases, PNA, PRNA, etc. (Genome Chemistry, Mitsuo Sekine, Retsu Saito, Kodansha Scientific Iku, 2003; Peptide nucleic acids, 2nd ed. PE Ni elsen, Horizon Bioscience 2004; WO 92/20702; W0 01/96355; WO 01/96356, etc.).

 A linker molecule means a molecule that can be bound to a carbon cluster via a covalent bond to form a linker. The linker molecule refers to the species before being attached to the carbon cluster, and the linker refers to the species after being attached to the carbon cluster. A linker molecule includes a reactive group or a protected form thereof capable of forming a covalent bond with an amino group on the carbon cluster and a reactive group or a protected form thereof capable of binding an organic molecule. A linker molecule usually has a structure in which a reactive group capable of forming a covalent bond with an amino group on a carbon cluster and a reactive group capable of binding an organic molecule are connected by a divalent organic group. Have.

 Examples of reactive groups that can form a covalent bond with an amino group of the carbon cluster include halogen (fluorine, chlorine, bromine and iodine), carboxyl group, hydroxyl group, active ester group, epoxy group, aldehyde group, and carposide imide. Group, imidazole group, isothiocyanate group, isocyanate group and the like.

The reactive group capable of bonding the structural unit of the organic compound to be synthesized is appropriately determined depending on the type of the organic molecule to be bonded. When synthesizing a polypeptide as an organic compound and binding a structural unit of an amino acid or a protected form thereof (for example, a protected lpoxyl group or an amino group) to a linker, the carboxyl of the amino acid A reactive group capable of forming a covalent bond with a group or amino group is preferred. Covalent bond with carboxyl group of amino acid Examples of the reactive group capable of forming an amino group include an amino group, a hydroxyl group, a carboxyl group, and a halogen (fluorine, chlorine, bromine and iodine). Examples of reactive groups capable of forming a covalent bond with the amino group of amino acid include force lpoxyl group, active ester group, epoxy group, aldehyde group, carpositimide group, imidazole group, isothiocyanate group, isocyanato group and the like. It is done. In the case of synthesizing a polynucleotide as an organic compound if it binds the a structural unit nucleoside or a protected form (e.g., those protecting the base of the Amino group) to the linker scratch, ³ nucleoside '- Reactive groups that form covalent bonds with hydroxyl groups are preferred. Examples of reactive groups that can form a covalent bond with a hydroxyl group include a carboxyl group, an amino group, a halogen (fluorine, chlorine, bromine and iodine), an epoxy group, an aldehyde group, and a carpositimide group. .

 A person skilled in the art can make a reactive group into a protected form by selecting an appropriate protecting group. For example, Harrison and Harrison, “Compendium of Organic Synthtic Methods”, 124-131, published by John Wiley and Sons, for protecting groups for each reactive group. See 1971. These protecting groups are removed during the reaction on the linker.

 The divalent organic group that binds the two reactive groups of the linker molecule or protected forms thereof is not particularly limited as long as it does not interfere with the target solid phase synthesis reaction. The organic group includes, for example, a divalent aliphatic group, a divalent cyclic group, or a divalent hydrocarbon group that is a combination of an aliphatic group and a cyclic group (including oxygen, nitrogen, sulfur, and silicon). It is OK.) Aliphatic groups include alkylene groups, alkenylene groups, and alkynylene groups. Cyclic groups include alicyclic groups, aromatic groups and heterocyclic groups. Here, the alicyclic group refers to a cyclic hydrocarbon group having characteristics similar to those of an aliphatic group, and the aromatic group refers to a mononuclear aromatic hydrocarbon group or a polynuclear aromatic hydrocarbon group. A heterocyclic group means a cyclic hydrocarbon group in which one or more atoms in the ring are substituted with an element other than carbon (such as oxygen, nitrogen, sulfur and silicon).

 The divalent organic group in one molecule of the linker preferably contains an aromatic ring. Including an aromatic ring facilitates separation from the organic compound carrier after solid-phase synthesis. Examples of the aromatic ring include a benzene ring, a phenanthrene ring, a fluorene ring, a naphthalene ring, an anthracene ring, and a pyrene ring.

 When the linker molecule is bonded to a carbon cluster, it becomes a linker having a reactive group capable of binding the structural unit of the organic compound to be synthesized or a protected form thereof.

 As a preferred linker molecule, the following formula I:

X— R ¹ — A— R ² — Y (I)

[Where

X is a reactive group capable of forming a covalent bond with the amino group of the carbon cluster or a protected form thereof, preferably halogen, more preferably fluorine. R ¹ is a direct bond or a substituted or unsubstituted divalent aliphatic group, preferably also properly unsubstituted unsubstituted C _I-6 alkylene or C ₂ - a ₆ alkenylene,

 A is a substituted or unsubstituted aromatic ring, preferably a substituted or unsubstituted funylene group, preferably a 1,4-phenylene group,

R ² is a direct bond or a substituted or unsubstituted divalent aliphatic group, preferably a substituted or unsubstituted alkylene or C _2-6 alkylene,

 Y is a reactive group capable of bonding a structural unit of an organic compound to be synthesized. When synthesizing a polypeptide, Y is preferably a hydroxyl group or a protected form thereof. When synthesizing a polynucleotide, , Preferably a carboxyl group or a protected form thereof]. Examples of the protecting group for the hydroxyl group include a acetyl group, a benzyl group, a silyl group, a tetrahydropyrenyl group, a methyl group, and a monomethoxymethyl group. Examples of the protecting group for the carboxyl group include groups capable of forming a strong rubonic acid derivative such as an ester, an acid halide, and an acid anhydride. Examples of the group capable of forming an ester include an alkoxy group, an aralkyloxy group, and an allyloxy group. Specific examples include a benzyloxy group and a t-butoxy group.

Examples of the substituent in R \ A and R ² include halogen, hydroxyl group, substituted or unsubstituted amino group, nitro group, cyano group, substituted or unsubstituted selected from fluorine, chlorine, bromine and iodine. C _w . An alkyl group, substituted or unsubstituted. Alkenyl group, substituted or unsubstituted. A cycloalkyl group, substituted or unsubstituted. Examples thereof include an alkoxy group, a substituted or unsubstituted alkoxycarbonyl group, and a carboxyl group.

 The aromatic ring represented by A ′, preferably F: the dilene group preferably has an electron-withdrawing substituent. Examples of the electron-withdrawing substituent include a nitro group, a nitroso group, a carbonyl group, a carboxyl group, a cyano group, a trialkylammonium group, and a trifluoromethyl group.

 In addition, a linker molecule that gives a benzyl alcohol functional group generally used in solid phase synthesis may be used (for example, Wang et al., J. Araer. Chem. Soc., 95, 1328 (1973) jo) Rink et al., Tetrahedron Letters, 28, 3787 (1987)).

 Specific examples of the linker molecule further include 2- (4-fluoro-2-nitrobenzyloxy) tetrahydropyran, funololonitronitrobenzene having polyethylene oxide, and succinic anhydride.

 When a linker molecule of the formula (I) forms a covalent bond with the amino group of the aminated carbon cluster, the following formula (II)

One NH— R ¹ — A— R ² — Y (II)

[Wherein NH— is bonded to carbon of carbon cluster and RA, R ² and Y are as defined for Formula I] A carrier for solid-phase synthesis formed by binding a linker represented by Prior to performing solid phase synthesis, the reactive group Y is usually in a protected form.

 In order to prevent side reactions in solid phase synthesis, it is preferable to protect the free amino group remaining on the carbon cluster after the linker molecule is reacted with the aminated carbon cluster of the present invention. The protecting group for the amino group is not particularly limited, and examples thereof include an acyl group, a carbamine group, a trialkylsilyl group, a phthalyl group, a carboxyalkylcarbonyl group, a tosino group, a trifluoroacetyl group, and a trityl group. , Penzinoreoxycarbonyl group, Fmoc (9-fluorenylmethoxycarbonyl) group, Boc (t-butoxycarbonyl) group, benzyloxycarbonyl group, Npys (3-nitro-2-pyridylsulfenyl) group and p-methoxybenzyl Examples include an oxycarbonyl group and a mono- or disubstituted trityl group.

 The solid phase synthesis method of the organic compound of the present invention includes sequentially reacting and binding the structural units of the organic compound to be synthesized using the carrier for solid phase synthesis of the present invention as a starting point.

 One embodiment in the case of solid-phase synthesis of a polypeptide as an organic compound will be described below. First, if the linker's reactive group (eg, hydroxyl or amino group) is protected, it is deprotected and covalently bonded to the carboxyl group of the first amino acid. Usually, the amino group of an amino acid is protected with a protecting group such as Fmoc group. After binding the first amino acid, the desired polypeptide is systematically synthesized. This synthesis is performed by repeated deprotection and coupling. The last protecting group on the amino acid attached can be subjected to an appropriate treatment to release the N-terminal amino group, for example by acid hydrolysis such as with trifluoroacetic acid in the case of the Boc group, or the Fmoc group. In this case, it is removed quantitatively by base treatment using piperidine. There are several ways to connect the N-terminus of the last amino acid attached to the C-terminus of the next amino acid. For example, it can be carried out with a condensation reagent such as, for example, dicyclohexylcal positive imide (Sheehan et al., J. Am. Chera. Soc., 1955, 77, 1067) or derivatives thereof, HATU, TBTU, HBTU.

 The condensation reaction between the polypeptide bound to the solid phase synthesis carrier of the present invention and the amino acid can be confirmed by a Kaiser test. Kaiser test: a) ethanol solution of 5% ninhydrin, b) 80 ° /. Ethanol solution in phenol, c) Add 5 drops each of 0.2 mM potassium cyanide pyridine solution and heat in boiling water for 5 minutes. If the solution is blue by this operation, the condensation reaction is continued. If the solution turns yellow, the amino group is deprotected and the condensation reaction with the next amino acid is performed. By repeating this cycle as many times as necessary, a polypeptide having the desired sequence and having a desired length can be obtained.

The final deprotection and release of the polypeptide from the carrier can be accomplished using strong acids such as anhydrous HF (Sakakibara et al., Bull. Chem. Soc. Jpn., 1965, 38, 4921), trifluoroacetic acid, tris (trifluoroacetic acid) boron (Pless et al., Helv. Chim. Acta, 1973, 46, 1609), sulfonic acids such as trifluoromethanesulfonic acid and methanesulfonic acid (Ya.iima et al., J Chem. Soc., Chem. Comm., 1974, 107), and a mixed solution of the above compound and cresol.

 Methods for solid-phase synthesis of polynucleotides are well known in the art, and for example, techniques such as phosphoramidite method, H-phosphonate method, triester method and the like can be used. As a starting point for solid phase synthesis, solid phase synthesis may be carried out according to a known method except that a carrier in which a single molecule of a linker is covalently bonded to an aminated carbon cluster is used.

 A synthesis method by the phosphoramidite method will be described below as an embodiment in the case of solid-phase synthesis of a polynucleotide. First, a nucleoside phosphite triester is obtained by condensing a nucleoside 3'-phosphoramidite with various functional groups protected and a nucleoside bonded to a carbon cluster via a linker. Next, after protecting the unreacted 5'-hydroxyl group, it is oxidized to obtain a nucleoside phosphate triester. Next, the 5′-hydroxyl protecting group of the chain extension product is removed to obtain a 5′-hydroxyl protected product. In the same manner, after extending the nucleotide chain to the target length in the same manner, all protecting groups are removed and the generated polynucleotide is separated from the carrier. As the protecting group for the nucleic acid base part, an acyl group such as a benzoyl group or an isobutylyl group can be used, and as the protecting group for the nucleotide part, a 2-cyanoethyl group can be used.

Unlike a general-purpose solid support, the aminated carbon cluster is physically strong and can be vigorously stirred, so that a solid-phase synthesis reaction can be carried out with high reaction efficiency. Furthermore, the present inventors have found that an analysis that cannot be realized with an existing resin carrier is possible by using an aminated carbon cluster with good dispersibility in a solvent as a carrier for solid phase synthesis. Since a normal resin solid support has a size of several hundreds of micrometers, it does not move freely when dispersed in a solvent. For this reason, it is difficult to directly detect the organic compound on the carrier. So far, a method has been developed to secure the mobility of the supported site by introducing a flexible PE0 chain into the linker site on the carrier and detect it with 丽 R. Its sensitivity and resolution are not comparable with solution spectra. Since the aminated carbon cluster 1 of the present invention is dispersed as a single particle in a solution and behaves as a huge molecule, it is presumed to have a mobility close to the free motion of a small molecule. The present inventors have found that an amino acid site on the support for solid phase synthesis of the present invention can be detected and identified by a solution ¹ H NMR spectrum. Until now, in order to analyze the reaction product obtained by the solid-phase synthesis method, it has been necessary to cleave from the support, and it has been complicated to confirm the success or failure of the reaction at each reaction stage. By using carbonized clusters, it is possible to confirm the success or failure of the reaction in each reaction stage on the support without cutting out the reaction product. Example

Example 1 Synthesis of hydrophilic aminated NHA 2 A mixture of carbon nanohorn aggregates (NHA) (200 mg) and sodium amide (200 rag) in liquid ammonia (320 mL) was refluxed in a flask equipped with a dry ice cooler for 3 hours. (-33 ° C). Removal of ammonia gave a black solid. The crude material was further washed with saturated NH ₄ C1 aqueous solution (100 mL), filtered and dried at 25 ° C, 0.2 mmHg for 12 hours to give aminated NHA 2 (205rag) black solid (Figure 1). Since NHA is highly absorbent, it was difficult to calculate the exact weight of aminated NHA 2. The reaction could easily be scaled up to an amount of 1.00 g NHA, yielding 1.03 g aminated NHA 2.

IR spectrum of raw NHA and aminated NHA 2 was obtained with ASI Applied Systems REACT IR1000 with attenuated total reflection (ATR) equipment (Figure 2) _ώ IR spectrum _{broadened to} 3220 cm- ¹ This indicates the presence of amino groups on NHA.

 The structure of aminated NHA 2 was analyzed using a transmission electron microscope (TEM; JEM2100F, JE0L) (Fig. 3A). Analysis by TEM showed that the conical structure of carbon nanohorn and the aggregated structure existed before and after the reaction.

Example 2 Amination Preparation and analysis of NHA 2 dispersion

 Amination NHA 2 (1.00 mg) was sonicated in water (5.00 mL) for 2 minutes (3.8 kHz; US-3, As One Co.) to give a clear gray solution ( (In Figure 3B). There were no visible particles in the solution, and no material adhered to the membrane (pore size 0.2 / im, Advantec) after filtration. The concentration of aminated NHA 2 can be further increased to 1.0 mg / mL or higher, but the solution becomes too dark to determine the presence of visible particles (Figure 3B). left). All the crude material obtained by the reaction was dissolved in water. This indicates that NHA was quantitatively converted to a hydrophilic form by the reaction.

 The size of the particles in the solution was analyzed by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern) (FIG. 3C). DLS studies have shown that aqueous solutions of aminated NHA 2 contain particles with an average size of 134 ± 5 nm. The size range is 70 ηπ! ˜500 nm, consistent with previous TEM results.

 The solution was deposited on My force and the particles were further analyzed by AFM (JSPM-4200, JE0L) (Figure 3D). Analysis by AFM showed that the particles had a spherical shape, and observation of well-isolated particles confirmed that NHA was present as a single particle in solution. Solid particle height analysis by AFM showed a smaller average size of 95 nm (Figure 4). This is thought to be due to the removal of the hydration layer under AFM conditions.

Example 3 Amination by Sanger's method Determination of amino groups on NHA

The amount of amino groups in aminated NHA 2 was proved and quantified using the method of Sanger (Encyclopedia of Reagents for Organic Synthesis Vol. 4, pp 2556-2557, Paquette, LA Eds. Wiley, 1995, Chichester) . That is, the amino group of aminated NHA 2 is converted to a dinitrophenylamino group with 1-fluoro-2,4-dinitrobenzene, and the amount is converted to UV-vis difference. Quantified by spectrum.

Amino 匕 NHA 2 (5.00 mg) was added to NaHC0 ₃ (0. 10 M, 100 μL) in basic aqueous solution and ethanol (50 i L) at 60 ° C for 20 min. , 4-dinitrobenzene (1.00 rag). The reaction mixture was filtered to give a black solid. The black solid was further washed with water (1.0 raL) and ethanol (1.0 mL) and dried under reduced pressure to obtain dinitrophenylaminated NHA 3 (5.28 mg) (Fig. 1).

The absorption spectrum of the aqueous solution of dinitrophenyl aminated NHA 3 (1.00 rag) had a peak corresponding to the dinitrophenyl group at 355 nm (FIG. 5B). Since none of the starting materials had an absorption at 355 nm, it was proved that an amino group was introduced into NHA by reaction with sodium amide. In addition, based on the reported molar absorption coefficient of dinitrophenylamino groups (16000-18000 M- ¹ · era), the amount of amino groups was calculated to be about 0.22 μmol / mg. NHA Since the surface area of γ is pre-calculated to be 308 ra ² / g (Murata,.; Kaneko, K .; Kokai, F .; Takahashi, K .; Yudasaka, M .; I ij ima, S. Chem . Phys. Lett. 2000, 331 , 14-20), ■ which shows the presence of one amino group of an area of 300 nm ^2.

 Example 4 Amination by column chromatography Fractionation of NHA 2

 One of the most difficult problems to standardize carbon particles is to obtain particles of uniform size. As a result of studying gel permeation chromatography for separation, the present inventors have found that a gel based on polyacrylamide (Sephacryl 500HR) is suitable as a medium. Therefore, an aqueous solution of aminated NHA 2 was eluted with a column of Cefacryl 500HR.

Pour ice solution of aminated NHA 2 (0.20 g / m 0.50 raL) into a column of Cefacryl 500HR (resin volume: 17 mL, column diameter: 1 cm, column height: 22 cm), 0.5 Elute with water at a flow rate of mL / min. A chromatogram was obtained using a UV detector (detected at 260 nm, UV-2075, JASC0) (Figure 6A) and the eluent was collected as a 0.25 raL fraction. The particle size of each fraction was analyzed by DLS (Figure 6B;). The fraction's 260 nra UV absorption showed that 92 ° / ₀ of the flowed particles eluted from the force ram.

 Also, the particles were fractionated by size, as shown in the typical data in FIG. 6B. The average particle size in fractions 22, 23, 24 and 27 were 198 ± 17, 148 ± 1, 134 ± 6 and 127 ± 7 nra, respectively.

 If gels with different pore sizes (Cefacryl 100HR or 1000SF) are used, the amount of eluted particles is 0% and 1. /. (Figure 7).

 Example 5 Synthesis of fluorescently labeled aminated NHA 4

Aminated NHA 2 (0. 40 rag / mL, 20 mL) in aqueous solution was added 2- (2, 7 difluoro-6-hydroxy-3-oxo-3H-xanthene-9-yl) -N- [5 -(2,5-Dioxopyrrolidine-1-yloxycarbonyl) -pentyl] -terephthalamidic acid (2.8 mg, Oregon Green 488-X, Invitrogen) A solution of ethanol (20 mL) was added and the mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered to give a black solid. The black solid was further washed with water (20 mL) and methanol (20 mL) and dried under reduced pressure to obtain fluorescently labeled aminated NHA 4 (8.23 mg) (FIG. 1). Aminated NHA 4 was analyzed by UV-vis difference spectrum (Fig. 5C). The absorption coefficient of the chromophore (84000 M " ¹ .cm- ¹ ) power, et al. The amount of Oregon Green 488 was calculated to be O. OU Ai mol / mg.

 When aminated NHA 2 was treated with Oregon Green 488 methyl ester under the same reaction conditions, the resulting NHA did not show absorption or excitation of fluorescent dyes (Figure 8). From this result, it was confirmed that the fluorescence of aminated NHA 4 was not due to the physically adsorbed dye but to the covalently bonded dye.

Example 6 Cytotoxicity test

Approximately 2xl0 ⁴ cells in 96-well plate in medium containing 10% fetal bovine serum, 1% penicillin and streptomycin (Dulbecco's modified Eagle's medium) at 37 ° C, humidified condition The cells were cultured at 24 hours. The culture medium was replaced with a new medium containing aminated NHA 2 at a rate of 0.05 mg / mL and cultured for 48 hours. Toxicity was calculated by lysing the cells, centrifuging, adding a protease inhibitor and Kumashi Brilliant Blue G-250, quantifying the protein from the absorption at 595 nm (Bradford method), and comparing with the control experiment. .

 An experiment similar to the above was performed with quartz particles and titanium dioxide particles. Aminated NHA 2 showed about one-tenth the toxicity of quartz particles, but compared with titanium dioxide particles that are generally non-toxic. And showed toxicity (Figure 9). In this example, the toxicity of nanotube particles whose structural characteristics were completely clarified was compared with that of existing particles. This example shows that the lyophilized carbon cluster prepared in the present invention can be used as a standard material for nanotube particles. Example 7 Mass synthesis of aminated carbon nanohorn aggregates

After striking Bonn Nanohorn aggregates (1.50 g) in liquid ammonia (2, 4 L), sodium amide (3.00 g) was added. The reaction mixture was stirred at reflux at −33 ° C. in a flask equipped with a dry ice condenser. After stirring for 6 hours, the ammonia was distilled off over 1 hour by gently heating in a 30 ° C hot water bath to obtain a black solid. The crude product was dispersed in saturated chloride ammonium Niumu solution (300 mL), after another Delo hydrophilic membrane filter (pore size 0. 2 _u m), the desired product remaining on the filter paper ultrapure water (300 mL ). Under reduced pressure (0.2 mmH _g ), the mixture was dried at room temperature for 12 hours to obtain an amino nanohorn aggregate as a black solid (1.52 g).

Example 8 Introduction of benzyl alcohol linkage (linker) moiety to amino nanohorn aggregates

(Black circle indicates carbon nanohorn) 4-Fluoro-3-nitrobenzene alcohol (l. OO g, 5.84 mraol), 3,4-dihydro-2H-pyran (0.690 mL, 7.60 difficult ol), Aluminum chloride hexahydrate (14.1 mg, 58.4 μπιοΐ) was stirred at 60 ° C., and returned to room temperature after 4 hours. The reaction mixture was purified directly by silica gel chromatography (eluent: methylene chloride). Drying at room temperature under reduced pressure (0.2mmHg) for 12 hours gave a pale yellow liquid of 2- (4-fluoro-3-nitrobenzyloxy) tetrahydropyran (1.45 g, 97%): IR (neat) 3070 ( w), 2944 (m), 2871 (m), 1622 (m), 1593 (m), .1538 (s), 1349 (s), 1254 (in), 1201 (m), 1123 (s), 1079 (ra), 1036 (in), 975 (ra), 906 (m), 833 (in), 818 (ra); 1H NMR '(500 MHz, CDC13) δ 1.56-1.68 (m, 4H), 1.75- 1.80 (ra, 2H), 1.85-1.89 (ra, 1H), 3.36-3.58 (ra, 1H), 3.85-3.89-(ra, 1H), 4.52-4.54 (d, J = 13 Hz, 1H), 4.71 -4.73 (t, J = 3.4 Hz, 1H), 4.79-4.82 (d, J = 13 Hz, 1H), 7.60-7.63 (ra, 1H), 8.1 (dd, J = 2, 4 Hz, 1H); 13C NMR (125 MHz, CDC1 ₃ ) δ 19.2 (CH ₂ ), 25.3 (CH ₂ ), 30.4 (CH ₂ ), 62.3 (C¾), 67.0 (CH ₂ ), 98.3 (CH), 118.2 (CH), 124.8 (C), 134.3 (CH), 135.7 (CH), 153.7 (C-Cl), 155.8 (C_N0 ₂ ); Anal Calcd for C ₁₂ H ₁₄ N0 ₄ F: C, 25.07; H, 5.53; N, 5.49. Found: C, 56.27; H, 5.68; N, 5.31.

 2- (4-Fluoro-3-nitrobenzyloxy) tetrahydropyran (13 mg, 5.5 匪 ol), potassium carbonate (750 mg, 5.5 thigh ol), amino nanohorn aggregates (504 rag; amount of amino groups: 0.11 ol) was mixed in DMF (25 raL) and stirred at 60 ° C for 20 hours. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 μϊα}, then washed with water (10 mL X 3), methanol (10 mL X 3), and toluene (10 mL X 3). Under reduced pressure (0.2 mmHg · 10 hours at room temperature) and further dried to obtain the desired product as a black solid (510 mg).

Example 9 Protection of residual amino group after introduction of benzyl alcohol linkage (linker) moiety

実施例 8で合成したリンカ一導入後のカーボンナノホーン （450 mg; ァミノ基の量： 12 μηιοΐ)とジメチルァミノピリジン （2.0 mg, 17 μηοϊ) を DMF (23 mL)中混合し、 この溶 液へ無水酢酸 （57 μ L, 0.61 ramol)、ジィソプロピルァミン （27 μ L， 0.15腿 ol)、 HOBt (1 - ヒドロキシベンゾトリァゾール， 2.5 mg, 18 μΐαοΐ) を加え、 室温下 2時間攪拌した。 反 応混合物を親水性メンブレンフィルター （孔径 0.2 μια) でろ過した後、 飽和炭酸水素ナ トリウム水溶液 （10 mL X 3)、 DMF (10 raL X 3)、 塩化メチレン （10 mL X 3)、 メタノ ール （10 mL X 3)で洗浄した。 減圧 （0.2 mmHg - 13 時間 .室温） 下、 さらに乾燥し、 残 存ァミノ基をァセチル化したカーボンナノホーンを黒色固体として得た （451 mg)。 実施例 1 0 ベンジルアルコール連結 （リンカ一） 都の THP保護基の除去

Carbon nanohorn (450 mg; amount of amino groups: 12 μηιοΐ) after introduction of the linker synthesized in Example 8 and dimethylaminopyridine (2.0 mg, 17 μηοϊ) were mixed in DMF (23 mL), and this solution was mixed. Add acetic anhydride (57 μL, 0.61 ramol), disopropylamine (27 μL, 0.15 ol), HOBt (1-hydroxybenzotriazole, 2.5 mg, 18 μΐαοΐ), and stir at room temperature for 2 hours. did. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 μια), then saturated aqueous sodium hydrogen carbonate (10 mL X 3), DMF (10 raL X 3), methylene chloride (10 mL X 3), methanol. (10 mL X 3). Under reduced pressure (0.2 mmHg-13 hours, room temperature), it was further dried to obtain carbon nanohorns having a remaining amino group acetylated as a black solid (451 mg). Example 1 0 Benzyl alcohol linkage (Linker I) Removal of THP protecting group in Tokyo

実施例 9で合成したリンカ一付きカーボンナノホーン （410 mg, THPの量： 76 /z mol)と p -トルエンスルホン酸マ水和物 （I0 mg， 58 μ οΙ) をメタノール （15 mL) 中混合し、 40°C で 4時間加熱攪拌した。 反応混合物を親水性メンブレンフィルター （孔径 0. 2 M m) でろ 過した後、 DMF (10 mL X 3)、 塩化メチレン (10 mL X 3)、 メタノール (10 mL X 3)で 洗浄した。 減圧 （0. 2讓 Hg . 12時間 '室温） 下溶媒を留去し、 目的生成物であるべンジル アルコール部位をもつカーボンナノホーンを黒色固体として得た （410 rag) ₀

Carbon nanohorn with linker (410 mg, amount of THP: 76 / z mol) synthesized in Example 9 and p-toluenesulfonic acid mahydrate (I0 mg, 58 μ οΙ) were mixed in methanol (15 mL). The mixture was heated and stirred at 40 ° C for 4 hours. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 M m), and then washed with DMF (10 mL X 3), methylene chloride (10 mL X 3), and methanol (10 mL X 3). The solvent was distilled off under reduced pressure (0.2 讓 Hg. 12 hours at room temperature), and the carbon nanohorn with the benzyl alcohol moiety as the target product was obtained as a black solid (410 rag) ₀

[Example 1] 'Introduction of Fmoc-protected glycine into the benzyl alcohol moiety (synthesis of N-Fmoc-Gly-HMP-AN)

この反応では DMFは'ニンヒドリン存在下で蒸留したものを用い、 溶媒中に残存するアミ ンを完全に除去した。 N- Fmoc グリシン （110 mg， 0. 36 mmol) を塩化メチレン （3· 0 mL)、 DMF (0. 5 mL) の混合溶媒中溶解し、 DIC (Ν, Ν' -ジイソプロピルカルポジイミド， 28 μ ΐ, 0. 18 ramol) を 0°Cで加え同じ温度で 20分間攪拌した。 溶媒を減圧留去した後得られた白 色固体に DMF (2. 5 mL) を加え、 再度溶解し、 この溶液とベンジルアルコール部をもつ力 一ボンナノホーン（52 mg， ヒドロキシル基の量： 95 mol)を混合し、 室温下 3時間攪拌し た。 反応混合物を親水性メンブレンフィルター （孔径 0. 2 μ πι) でろ過した後、 DMF (10 mL X 3)、 塩化メチレン （10 mL X 3)、 メタノール （10 mL X 3)で洗浄した。 減圧 （0. 2 mmHg · 12時間'室温） 下、 さらに乾燥し、 Fmoc保護グリシンと結合したカーボンナノホーンを黒 色固体として得た （50 mg)。 合成した Fmoc保護グリシン 'カーボンナノホーン複合体 1. 0 ragを重 DMF (0. 5 mL) 中に分散し、 醒 Rを測定することで Fmocグリシン部おょぴベン ジルアルコールリンカ一部の信号を検出、 同定した （図 1 0 )。

In this reaction, DMF was distilled in the presence of ninhydrin to completely remove the residual amine in the solvent. N-Fmoc Glycine (110 mg, 0.36 mmol) is dissolved in a mixed solvent of methylene chloride (3.0 mL) and DMF (0.5 mL), and DIC (Ν, Ν '-diisopropylcarposimide, 28 μ 0, 0.18 ramol) was added at 0 ° C and stirred at the same temperature for 20 minutes. DMF (2.5 mL) was added to the white solid obtained after distilling off the solvent under reduced pressure, and then dissolved again. This solution and benzyl alcohol part had a strong bon nanohorn (52 mg, amount of hydroxyl group: 95 mol) were mixed and stirred at room temperature for 3 hours. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 μπι), and then washed with DMF (10 mL X 3), methylene chloride (10 mL X 3), and methanol (10 mL X 3). Further drying was performed under reduced pressure (0.2 mmHg · 12 hours at room temperature) to obtain carbon nanohorn combined with Fmoc-protected glycine as a black solid (50 mg). The synthesized Fmoc-protected glycine 'carbon nanohorn complex 1.0 rag is dispersed in heavy DMF (0.5 mL), and the signal of the Fmoc glycine part opi-benzyl alcohol linker is obtained by measuring the arousal R. Detected and identified (Fig. 10).

¾ NMR (400 MHz, DMF— d ₇ ) δ 3.69 (s, 1Η), 3. 89 (m, 2H), 3.94 (m, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7. 30-7. 38 (br ra, 2H), 7. 42-7. 45 (br ra, 2H), 7. 64 (d, J = 7.6 Hz, 1H), 7 70-7.75 (br m, 1H), 7. 78-7.93 (br ra, 1H). Example 1 2 Introduction of Fmoc retention alanin into the benzyl alcohol moiety (synthesis of N-Fmoc-Ala-HMP-A)

 .

 In this reaction, DMF distilled in the presence of ninhydrin was used to completely remove the amine remaining in the solvent. N-Fmoc-L-alanine (110 mg, 0.36 ol) is dissolved in a mixed solvent of methylene chloride (3.0 mL) and DMF (0.5 mL), and DIC (Ν, Ν '-diisopropyl carbonate) is dissolved. Diimide, 28 μΐ, 0.18 olol) was added at 0 ° C and stirred for 20 minutes at the same temperature. DF (2.5 mL) was added to the white solid obtained after distilling off the solvent under reduced pressure, and the solution was dissolved again. This solution and benzyl alcohol part had a strong bonbon horn (51 mg, amount of hydroxyl groups: 95 μ ιηοΐ) were mixed and stirred at room temperature for 3 hours. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 μηι), and then washed with DMF (10 mL X 3), methylene chloride (10 mL X 3), and methanol (10 mL X 3). Further drying was performed under reduced pressure (0.2 mmHg · 12 hours. Room temperature) to obtain carbon nanohorn bound to Fmoc-protected glycine as a black solid (53 mg). The synthesized Fraoc-protected alanine 'carbon nanohorn complex (1.0 mg) was dispersed in heavy DMF (0.5 mL), and 丽 R was measured to detect signals from the Fmoc alanine and benzyl alcohol linkers. Was detected and identified (Fig. 11).

¾ 丽 R (400 MHz, DMF-d ₇ ) δ 1. 05 (d, J = 6.9 Hz, 3H), 3.68 (s, 1H), 4.11--4.12 (m, 2H) , 4. 27 (m, 1H), 7. 10 (d, J = 7.6 Hz, 1H), 7. 34—7. 38 (br ra, 2H), 7. 42-7. 45 (br m , 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.76 (br ra, 1H), 7. 78-7.98 (br m, 1H).

Example 1 3 Introduction of Fmoc-protected valine into benzyl alcohol (synthesis of N-Fmoc-Val-HMP-AN)

この反応では DMFはニンヒドリン存在下で蒸留したものを用い、 溶媒中に残存するアミ ンを完全に除去した。 N- Fmoc- L-パリン （120 mg, 0. 36 mraol) を塩化メチレン （³. O mL；)、 DMF (0. 5 mL) の混合溶媒中溶解し、 DIC (N, N' -ジイソプロピルカルポジイミド， 28 μ ΐ, 0. 18 ramol) を 0°Cで加え同じ温度で 20分間攪拌した。 溶媒を減圧留去した後得られた白 色固体に DMF (2. 5 mL) を加え、 再度溶解し、 この溶液とベンジルアルコール部をもつ力 一ボンナノホーン （51 mg, ヒドロキシル基の量： 95 mol) を混合し、 室温下 3時間攪拌 した。 反応混合物を親水性メンブレンフィルター （孔径 0. 2 _M ra) でろ過した後、 DMF (10 mL X 3)、 塩化メチレン （10 raL X 3)、 メタノール （10 mL X 3)で洗浄した。 減圧 (0. 2 raraHg · 12時間'室温） 下、 さらに乾燥し、 Fmoc保護グリシンと結合したカーボンナノホー ンを黒色固体として得た （50 mg)。 合成した Finoc保護パリン'カーボンナノホーン複合体 1. 0 mgを重 DMF (0. 5 raL) 中に分散し、 匪 Rを測定することで Fmocバリン部およびべ ンジルアルコールリンカ一部の信号を検出、 同定した。

In this reaction, DMF distilled in the presence of ninhydrin was used to completely remove the amine remaining in the solvent. N-Fmoc-L-heparin (120 mg, 0. 36 mraol) methylene chloride ^{(. 3 O mL;),} DMF was dissolved in a solvent mixture of (0. 5 mL), DIC ( N, N '- diisopropyl Cal Po Diimide, 28 μΐ, 0.18 ramol) was added at 0 ° C and stirred for 20 minutes at the same temperature. White obtained after distilling off the solvent under reduced pressure Add DMF (2.5 mL) to the colored solid, dissolve it again, and mix this solution with bonbon nanohorn (51 mg, amount of hydroxyl group: 95 mol) with benzyl alcohol. Stir at room temperature for 3 hours. did. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 _M ra), and then washed with DMF (10 mL X 3), methylene chloride (10 raL X 3), and methanol (10 mL X 3). Further drying under reduced pressure (0.2 raraHg · 12 hours at room temperature) yielded carbon nanohorn bound to Fmoc-protected glycine as a black solid (50 mg). Disperse 1.0 mg of the synthesized Finoc-protected palin 'carbon nanohorn complex in heavy DMF (0.5 raL) and measure 匪 R to detect signals from the Fmoc valine part and benzyl alcohol linker. Identified.

lH NMR (400 MHz, DMF— d ₇ ) δ 1.00 (d, J = 6.4 Hz, 6H), 2.25 (br m, 1H), 4.1 1-4. 12 (m, 2H) , 4. 29 (m, 1H), 7. 10 (d, J = 7.6 Hz, 1H), 7. 30-7.38 (br m, 2H), 7. 42-7. 45 (br ra , 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.76-7.77 (br ra, 1H), 7, 78-7.98 (br ra, 1H).

Example 1 4_Removal of protecting group from Fraoc protected glycine on carbon nanohorn

Carbon nanohorn (30 mg) linked with Fmoc-protected glycine was dispersed in 50% piperidine / DMF (1.5 raL) and stirred at room temperature for 1 hour. The reaction solution was filtered through a hydrophilic membrane filter (pore size 0.2 μια), and then washed with DMF (10 raL X 3), methylene chloride (10 mL X 3), and methanol (10 mL X .3). Under reduced pressure (0.2 raraHg · room temperature · 12 hours), it was further dried to obtain a glycine-carbon nanohorn complex from which the Fmoc protecting group had been removed (30 rag). The N- (9-fluorenylmethyl) piperidine obtained in the filtrate was concentrated and purified by silica gel column chromatography (eluent: 1: 1 ethyl acetate / chloroform form) as a white solid. Obtained (1.5 mg, 5.7 rag ₀

Example 15 Removal of protecting groups from Fmoc protected alanine on carbon nanohorn

Carbon nanohorn (30 rag) linked with Fmoc-protected alanine was dispersed in 50% piperidine / DMF (1.5 raL) and stirred at room temperature for 1 hour. The reaction mixture was filtered through a hydrophilic membrane filter (pore size 0.2 μηι), and then washed with DMF (10 mL X 3), methylene chloride (10 mL X 3), and methanol (10 raL X 3). Further drying under reduced pressure (0.2 mmHg · room temperature · 12 hours) yielded an alanine · carbon nanohorn complex from which the Fmoc protecting group was removed as a black solid (29 in _g ). In the filtrate The resulting N- (9-fluorenylmethyl) piperidine was concentrated and purified by silica gel chromatography (eluent: 1: 1 ethyl acetate / chloroform) to obtain a white solid ( .1. 5 mg, 5.7 mg _D

 Example 1 6 Removal of protecting group from Fmoc protected palin on carbon nanohorn

Carbon nanohorn (30 mg) linked with Fmoc-protected valine at 50 ° /. The mixture was dispersed in piperidine / DMF (1.5 mL) and stirred at room temperature for 1 hour. The reaction solution was filtered through a hydrophilic membrane filter (pore size 0.2 m), and then washed with DMF (10 mL X 3), methylene chloride (10 mL X 3), and methanol (10 mL X 3). Under reduced pressure (0.2 ramHg · room temperature · 12 hours), the product was further dried to obtain a valine · carbon nanohorn complex from which the Fmoc protecting group was removed as a black solid (29 mg;). The N- (9-fluorenylmethyl) piperidine obtained in the filtrate was concentrated and purified by silica gel column chromatography (eluent: 1: 1 ethyl acetate / chloroform). It was obtained as a white solid _{(1. 3 m g, 4. 9} mg). All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

The scope of the claims 1. A hydrophilic carbon cluster having a structure in which a substituted or unsubstituted amino group is bonded to a carbon atom of the carbon cluster. 2. The hydrophilic carbon cluster according to claim 1, wherein the amino group is an unsubstituted amino group. 3. The hydrophilic carbon cluster according to claim 1 or 2, wherein the carbon cluster is a tubular carbon cluster.  4. The hydrophilic carbon according to claim 3, wherein the carbon cluster is carbon nanohorn, and the substituted or unsubstituted amino group is bonded to a carbon atom of at least a conical end cap. 'Elemental cluster. 5. A carbon cluster dispersion in which the hydrophilic carbon cluster according to any one of claims 1 to 4 is dispersed in an aqueous solvent. 6. A method for producing a hydrophilic carbon cluster having a structure in which a substituted or unsubstituted amino group is bonded to a carbon atom of a carbon cluster, which comprises reacting the carbon cluster with a metal amide. 7. The method of claim 6, wherein the carbon cluster is a tubular carbon cluster. 8. The method according to claim 6 or 7, wherein the metal amide is an alkali metal amide. 9. The method according to any one of claims 6 to 8, further comprising chemically modifying an amino group bonded to a carbon atom of the carbon cluster. 10. A carrier for use in solid phase synthesis of an organic compound comprising the hydrophilic carbon cluster according to any one of claims 1 to 4. 11. A carrier for use in solid phase synthesis of organic compounds, The carrier according to any one of claims 1 to 4, wherein the carrier has a structure in which a linker molecule for solid-phase synthesis is covalently bonded to the amino group of the hydrophilic carbon cluster according to any one of claims 1 to 4. 12. A method for solid-phase synthesis of an organic compound using the carrier according to claim 10 or 11. 13. The method according to claim 12, wherein the organic compound is a polypeptide or a polynucleotide.