JP2011178756A - Polyion dendrimer for intracellularly introducing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polyion dendrimer capable of intracellularly transporting a protein by such a simple operation as to merely make mixing through utilizing intermolecular interaction via no covalent bond, thus useful as a highly versatile protein transporter, and to provide a method for transporting such a protein in high efficiency using the same. <P>SOLUTION: The polyion dendrimer is provided, being characterized by including cationic groups selected from guanidine groups, thiourenium groups and isothiourenium groups on its surface, having polyalkyleneoxy groups as branched chains, and also in that benzophenone group-bearing substituents are bound to the core part. A protein transporter using the polyion dendrimer, and a method for transporting a protein using the transporter, are also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention has a cationic group selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group as a surface portion, a polyalkyleneoxy group as a branched chain, and a benzophenone group as a core portion. The present invention relates to a polyion dendrimer in which a substituent is bonded. The polyion dendrimer of the present invention is useful as a highly versatile protein transport agent that can transport proteins into cells by simple operation of mixing using intermolecular interaction without using a covalent bond. The invention further relates to a protein transport agent using the polyion dendrimer of the present invention, and a method for transporting the protein into living cells.

Protein is attracting attention as a new type of drug because of its functional specificity and high biocompatibility. In general, polymer compounds such as proteins have a problem of extremely low cell membrane permeability. Therefore, it is essential to use a carrier that enables efficient intracellular introduction.
As one approach for realizing intracellular protein uptake, a method of linking intracellular transport peptides containing a large number of arginines is known (see Non-Patent Document 1). However, in this method, since it is necessary to link the peptide to the target protein by a covalent bond, there is a problem that the protein may be denatured or the function may be lost.

  As another approach, a noncovalent method using a cationic lipid has been proposed (see Non-Patent Document 2). This method is a method in which a cationic protein forms a micelle that encapsulates a protein, and the micelle is fused with a cell membrane to transfer a target protein into the cell. In this method, unlike the method using the intracellular transport peptide described above, since no covalent bond formation is required, denaturation and inactivation of the target protein can be avoided. However, since protein transport using cationic lipids requires lipid molecules at a concentration of 10 μM or more, sufficient transport efficiency could not be realized in vivo, which is diluted by the influence of diffusion. For this reason, the development of a carrier that can efficiently transport a protein into a cell as it is in vivo has been awaited.

Dendrimer is a dendritic polymer with a structure that is regularly branched from the center, named after a combination of the Greek words “dendri-” (dendritic) and “meros” (partial), and the structure is precisely controlled. Dendritic polymer. Over 5,000 papers have been published in the past decade, but it is considered difficult to put into practical use because it is extremely difficult to synthesize compared to other polymers. PAMAM dendrimers having a polyamidoamine structure, which are most frequently used at present, have already been marketed.
A dendrimer consists of a central molecule called the core, a dendritic branched structure called the dendron, and an end group called the surface. The number of branches of the dendron is generated. they said. In general, macromolecules have a certain molecular weight distribution, but high-generation dendrimers have the distinctive feature that even if they have a molecular weight of tens of thousands, they have almost a single molecular weight.
The core is said to determine the size, shape, orientation, and diversity of the entire dendrimer. The dendron is a part where the number of branch-like cells increases regularly. Determines the type and size of the space, and the multiplicity of the branch cells increases exponentially with generation. The surface is composed of reactive and non-reactive end groups, can express various functions depending on the type of end groups, and also plays a role of a gate that controls the entrance and exit of external guest molecules. .

Methods for producing dendrimers are well known, for example, methods for producing dendrimers based on a layer of lysine units (see Patent Document 1), methods for producing dendrimers and PAMAM dendrimers based on other units containing polyamidoamine (Patent Document 2). For example). These dendrimers are considered suitable for use as surface modifiers, metal chelators, demulsifiers or oil / water emulsions, wet paper strength enhancers in papermaking, and viscosity modifiers in aqueous formulations such as paints. However, the use as a base for pharmaceutical production (see Patent Document 3) and the association with a carrier substance that can be a biological reaction modifier (see Patent Document 4) have already been reported. Yes.
In addition, dendrimers (dendritic polymer compounds) having a cationic group such as a guanidine group as a terminal group on the surface are also known. For example, surfaces such as polyamidoamine dendrimers, polylysine dendrimers, and poly (propyleneimine) dendrimers are known. A dendrimer having an ionic group such as a quaternary amino-containing moiety, a pyridinium-containing moiety, a guanidinium-containing moiety, or an amidinium-containing moiety for the prevention or treatment of a disease caused by a toxic substance (see Patent Document 5) or the same dendrimer Those used for prevention or treatment of diseases caused by microorganisms such as bacteria or parasites (see Patent Document 6), and those using dendrimers having such ionic end groups as gel forming agents as carriers for agricultural chemicals and hygiene products (See Patent Document 7) and the like have been reported. Furthermore, a method has also been reported in which a cationic substance such as polyethyleneimine dendrimer having a cationic group is combined with an anionic enzyme-active fluorescent substrate to form a membrane transport system of the fluorescent substrate (see Patent Document 8).
However, it is not known that a substituent having a benzophenone group is bonded to the core.

U.S. Pat. No. 4,289,872 US Pat. No. 4,587,329 U.S. Pat. No. 4,410,688 International Patent Publication No. WO95 / 24221 JP-T-2002-524523 Special table 2002-524524 gazette Special Table 2002-538186 JP 2006-517382 A

Shiroh Futaki, Membrane-permeable arginine-rich peptides and the translocation mechanisms, Advanced Drug Delivery Reviews 57 (2005) 547-558 B.T.F.van der Gun et al, Serum insensitive, intranuclear protein delivery by the multipurpose retained lipid SAINT-2, Journal of Controlled Release 123 (2007) 228-238

  A problem to be solved by the present invention is a highly versatile protein transporter that can transport proteins into cells by simple operation of mixing using intermolecular interaction without using a covalent bond. It is an object to provide a polyion dendrimer useful as a protein transport agent, a protein transport agent using the polyion dendrimer, and a highly efficient transport method for proteins. Another goal is to establish design guidelines and synthesis methods for molecules that serve as the basic skeleton of such protein transport agents.

  The inventors focused on the particularly strong interaction between a plurality of ionic species, and designed the molecular design of a polycation dendrimer having many guanidine groups or thiourenium groups known as side chains of arginine on the molecular surface. We have been trying to explore the possibility of more efficient protein transport agents by establishing synthetic methods. Because these compounds have multiple ionic functional groups on the surface, they can be expected to have high cell membrane permeability and also serve to strongly bind to biomolecules with multiple anionic functional groups such as target proteins and nucleic acids. It can be expected that a protein-carrier complex can be formed at a low concentration. Furthermore, we found that the hydrophobic benzophenone group linked to the core has the effect of accumulating and concentrating the protein-carrier complex on the cell membrane by actively entering the hydrophobic layer in the cell membrane. The present invention has been reached.

  That is, the present invention has a cationic group selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group as a surface portion of a dendrimer, a polyalkyleneoxy group as a branched chain, and a core. The present invention relates to a polyion dendrimer in which a substituent having a benzophenone group as a part is bonded. More specifically, the present invention relates to the following general formula [1]

[Wherein X represents the following general formula [2] or [3]

[Wherein, k represents 0 or an integer of 1 to 5. ]

[Wherein, k represents 0 or an integer of 1 to 5. ]
And Y represents a cationic group independently selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group, and l represents 1-6. Represents an integer, m represents 0 or 1, n represents the number of generations, and represents an integer of 1 to 5; The branched 3,4,5-trihydroxybenzoic acid amide in the general formula [1] may be 3,4,5-trihydroxycinnamic acid amide, and the branched ethyleneoxy chain is It may be a linear or branched alkylene chain having 1 to 6 carbon atoms, preferably 2 to 5 carbon atoms. ]
The polyion dendrimer represented by these.
More specifically, the present invention relates to the following general formula [4]

[Wherein Y represents a cationic group independently selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group, and m represents 0 or 1. ]
The polyion dendrimer represented by these.
More specifically, the present invention relates to a polyion dendrimer represented by the following formula [5].

The present invention also relates to a protein transporter or a composition for protein transporter comprising the polyion dendrimer of the present invention described above.
Furthermore, the present invention relates to a method for transporting a protein into a biological cell membrane using the above-described protein transport agent or composition for protein transport agent comprising the polyion dendrimer of the present invention.

The present invention will be described in detail as follows.
(1) The dendrimer has a cationic group selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group, a polyalkyleneoxy group as a branched chain, and a benzophenone in the core portion. A polyion dendrimer, wherein a substituent having a group is bonded.
(2) The above (1), wherein the branched chain is a polyethyleneoxy group having 1 to 6 repetitions, which may have a 1,2,3-triazole group bonded to the terminal at the 1,4-position. Polyion dendrimer.
(3) The polyion dendrimer according to (1) or (2) above, wherein the branched portion is composed of 3,4,5-trihydroxybenzoic acid amide or 3,4,5-trihydroxycinnamic acid amide. .
(4) The polyion dendrimer according to any one of (1) to (3), wherein the substituent having a benzophenone group is a substituent having a benzophenone group represented by the general formula [2] or [3]. .
(5) The polyion dendrimer according to any one of (1) to (4), wherein the generation is 1 to 5 generations.
(6) The polyion dendrimer according to any one of (1) to (5), wherein the polyion dendrimer is a polyion dendrimer represented by the general formula [1].
(7) The polyion dendrimer according to any one of (1) to (6), wherein the polyion dendrimer is a polyion dendrimer represented by the general formula [4].
(8) The polyion dendrimer according to any one of (1) to (7), wherein the polyion dendrimer is a polyion dendrimer represented by the formula [5].
(9) A protein transporter comprising the polyion dendrimer according to any one of (1) to (8).
(10) A protein transporting method comprising using the protein transporting agent according to (9).

The polycation type dendrimer (dendritic polymer) of the present invention can form a protein-carrier complex by a strong intermolecular interaction with a target protein without using a covalent bond, and can be rapidly formed at a low concentration. In addition, the protein can be transported into the cell without impairing the activity of the protein. In addition, the protein transport activity maintains sufficiently high efficiency even in the presence of serum, and exhibits sufficient activity in vivo.
Furthermore, the polycation type dendrimer (dendritic polymer) for protein carrier of the present invention does not show cytotoxicity even at a practical concentration of 1 μM.

As described above, the polycation type dendrimer having a large number of cationic groups such as guanidine of the present invention interacts strongly with a protein, and as a protein transport agent to cells, a conventional cationic lipid type protein introduction reagent SAINT- It is possible to achieve high-efficiency protein introduction into cells that far exceeds phD. Since the polycation type dendrimer (dendritic polymer) of the present invention can be used quickly and safely at a low concentration as compared with conventional reagents, it can be applied in vivo, which has been difficult with conventional ones. It becomes possible.
In addition, the present invention molecularly designs a dendritic polymer having a cationic group such as a guanidine group as a surface portion, a highly hydrophilic polyethylene glycol group as a branched chain, and a substituent having a benzophenone group as a core portion, A manufacturing method was established.

FIG. 1 shows the results of comparing protein uptake activities by dendrimer G1-BP of the present invention (right end of FIG. 1), Arg _9, G1-NOMe, G1-NFITC, and SAINT-phD using fluorescently labeled actin as a protein. Is shown. The left end of FIG. 1 is a control, and the right side is a case where only actin is contacted. FIG. 2 shows the results of observation of protein uptake by the dendrimer G1-BP of the present invention with a confocal microscope. FIG. 3 shows the results of analyzing the effect of serum on the protein uptake activity by the dendrimer G1-BP or SAINT-phD of the present invention. FIG. 4 shows the results of analyzing the influence of contact time and concentration on the protein uptake activity by the dendrimer G1-BP of the present invention. FIG. 5 shows the result of observing with a confocal microscope whether the activity of the protein taken up by the dendrimer G1-BP of the present invention is maintained. FIG. 6 shows the result of analyzing the cytotoxicity of the dendrimer G1-BP of the present invention.

Hereinafter, the present invention will be described in more detail.
The feature of the polyion dendrimer of the present invention is that it first has a cationic group selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group on the surface of the dendrimer. Examples of such a cationic group include the following formulas [6], [7], and [8],

Is mentioned. The hydrogen atom portion of these groups may be substituted with a linear or branched alkyl group having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, such as a methyl group, an ethyl group, or a propyl group.
One or more such cationic groups may be present on the surface of the dendrimer. Preferably, all of the terminals of the surface portion have these cationic groups.
Further, such cationic groups on the surface of the dendrimer may be the same or different, but it is preferable that they are all the same from the viewpoint of ease of production.
Recently, attention has been focused on the action of arginine, an amino acid having a guanidine group. In particular, it has been reported that a compound into which arginine is introduced increases the cell membrane permeability, and this reason is attributed to a cationic group such as a guanidine group. Also in the dendrimer (dendritic polymer) of the present invention, it is considered that one of the reasons why the dendrimer of the present invention has excellent cell membrane permeation is that it has many cationic groups such as guanidine groups.

A feature of the polyion dendrimer of the present invention is that it has a highly hydrophilic polyalkyleneoxy group as a branched chain. Examples of the alkylene group in the present invention include linear or branched alkylene groups having 1 to 6 carbon atoms, preferably 2 to 5 carbon atoms. Preferred alkyleneoxy groups include ethyleneoxy groups and propyleneoxy groups, but ethyleneoxy groups are preferred from the standpoint of availability and hydrophilicity. Further, these alkyleneoxy groups may be substituted with a hydrophilic group such as a hydroxyl group or an alkoxy group having 1 to 5 carbon atoms.
Although there is no restriction | limiting in particular as the repeating number of the alkyleneoxy group in a branched chain, Preferably it is 1-10, More preferably, it is 1-6, More preferably, 2-4 is mentioned. Moreover, in each generation of dendrimers, the number of these repetitions may be the same or different. These repeat numbers ensure a large cell space inside the dendrimer, so a relatively large repeat number is required when introducing a large group having a hydrophobic benzophenone group in the core. If the group having a hydrophobic benzophenone group is small, the number of repetitions may be relatively small.
In the general formula [1] of the present invention, an ethyleneoxy chain is exemplified as the branched chain. As described above, the branched chain of the polyion dendrimer of the present invention is exemplified in the general formula [1]. The chain is not limited to the ethyleneoxy chain, and may be any linear or branched alkylene chain having 1 to 6 carbon atoms, preferably 2 to 5 carbon atoms, as long as it is a highly hydrophilic polyalkyleneoxy chain. Good.

The third feature of the polyion dendrimer of the present invention is that a substituent having a carboxyl group as a core portion and a benzophenone group is bonded to the carboxyl group via an alkyleneoxy group. The carboxyl group may be derived from any compound as long as it has a group capable of forming a branched chain as long as it can exist as a free carboxyl group. However, those derived from the same compound as the molecule forming the branched portion in the dendrimer of the present invention are preferable. For example, polyhydroxycarboxylic acid, polyaminocarboxylic acid, tricarboxylic acid, tetracarboxylic acid and the like can be mentioned. Preferred examples include 3,4,5-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid amide, 3,4,5-trihydroxycinnamic acid, or 3,4,5-trihydroxycinnamic acid. Examples include acid amides.
In general formulas [1], [4], and [5], 3,4,5-trihydroxybenzoic acid amide is exemplified, but the present invention is not limited thereto.
Moreover, there is no restriction | limiting in particular as the repeating number of the alkyleneoxy group couple | bonded with the said carboxy group, Preferably it is 2-7, More preferably, it is 2-5, More preferably, 3-5 is mentioned. The benzophenone group may be directly bonded to the alkyleneoxy group bonded to the carboxyl group, or may be bonded through, for example, a thiourea group. As a preferable example,

As shown in Formula [9] and Formula [10], a functional group for covalently bonding to a carboxy group can be introduced into benzophenone to form an amide bond with the carboxy group in the core part.
The bond between the carboxyl group in the core and the substituent having benzophenone may be in any form as long as a covalent bond is possible. Preferred examples of the bond include an amide bond and an ester bond.
The benzophenone in the substituent having a benzophenone group is a linear group having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, in addition to a functional group for bonding to the carboxy group of the core part, for example, a functional group such as a hydroxyl group. It may be substituted with an inert group such as a branched alkyl group. Further, the position of the functional group of benzophenone for binding to the carboxy group of the core part is not particularly limited, and may be any of 2-6 positions. A preferred position is the 4th position.

The compound that forms the branched portion of the polyion dendrimer of the present invention is not particularly limited as long as it can be branched, but the use of the same type of compound as the above-mentioned core portion compound is preferable. For example, when 3,4,5-trihydroxybenzoic acid or 3,4,5-trihydroxycinnamic acid is used as the compound having a carboxy group in the core portion, it can be used even in the branched portion. It is also preferable from the viewpoint of ease.
The bond between the branched portion compound and the branched chain can also be arbitrarily selected. For example, any bond such as an ester bond, an amide bond, an ether bond, and an imino (—N—) bond can be selected. Moreover, a triazolylmethyl group can also be inserted as needed.

As a method for producing the polyion dendrimer of the present invention, a production method according to a known production method for dendrimers can be applied. For example, the core part and the first generation are manufactured by the method of forming the core part and manufacturing the first generation, or the method of forming the core part in the state where the first generation is formed, and then, if necessary, The method of growing with the 2nd generation and the 3rd generation is mentioned.
And it can manufacture by forming the terminal part of a surface.
More specifically, refer to the examples described later.

In the polyion dendrimer of the present invention represented by the general formula [1], the branched chain is an ethyleneoxy group, the branched portion is 3,4,5-trihydroxybenzoic acid, and the branched chain and amide of the previous generation are used. It is connected by a bond, and it is connected to the next generation by an ether bond, and the number of branch repeats is 1 and the generation is n.
Further, the above general formula [4] represents the polyion dendrimer of the present invention in one generation state.
These are all examples of preferred polyion dendrimers of the present invention. More specifically, as preferred polyion dendrimers of the present invention,

The polyion dendrimer (Hereinafter, this compound is called G1-BP.) Represented by these.

The polyion dendrimer of the present invention not only has cell membrane permeability but can also associate with a protein to form a protein-carrier complex. Since the polyion dendrimer of the present invention is only associated with a protein and is not covalently bound, the protein structure is maintained in the cell membrane while maintaining the protein activity without any change in the structure of the protein. Can be introduced. Accordingly, the present invention provides a protein-carrier complex of the polyion dendrimer of the present invention and a protein.
The present invention provides a protein transporter into cells comprising the polyion dendrimer of the present invention. The protein transporting agent of the present invention can transport the target protein into cells quickly, safely and efficiently at a concentration of 0.01 μM or more, preferably 0.05 to 5 μM. In addition, the protein transporter of the present invention is less susceptible to serum and can be used in vivo. The present invention also provides a protein transport carrier (carrier) into cells comprising the polyion dendrimer of the present invention. Furthermore, the present invention provides a composition for transporting proteins into cells comprising the polyion dendrimer of the present invention and a solvent for transporting proteins.
The present invention provides a method for transporting a protein, characterized by using the protein transport agent of the present invention. The transport method of the present invention is performed by mixing the target protein and the protein transport agent of the present invention, appropriately diluting the obtained mixture with a buffer solution or a culture solution, and bringing the mixture into contact with target cells. Can do. According to the transport method of the present invention, a target protein can be transported to a target cell quickly, safely and efficiently.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

Production of G1-BP The desired dendrimer G1-BP of the present invention was produced according to the chemical reaction formula shown below.

(1) Production of Compound 3 1.66 g (5.04 mmol) of p-toluenesulfonic acid 2- (2- (2-azidoethoxy) ethoxy) ethyl (Compound 1) and 1.00 g of 4-hydroxybenzophenone (Compound 2) (5.04 mmol) was dissolved in 15 ml of dimethylformamide (DMF). 2.09 g (15.1 mmol) of potassium carbonate was added, and the mixture was stirred at 80 ° C. for 18 hours. After dilution with chloroform, the solid was removed by filtration, and the solvent was removed at 60 ° C. under reduced pressure. The obtained solid was dissolved in ethyl acetate, washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. After removing the solvent, the residue was purified by column chromatography (alumina, chloroform), (silica gel, chloroform) twice to obtain compound 3 as a light yellow oily transparent liquid (1.60 g; yield 89%).
¹ H-NMR (CDCl ₃ ): δ (ppm)
3.37 (t, 2H; CH ₂ N ₃ ), 3.63-3.80 (m, 6H; OCH ₂ ),
3.90 (t, 2H; Ar- OCH 2 CH 2), 4.21 (t, 2H; Ar-OCH 2),
6.98 (d, 2H; 3,5-Ar-H), 7.41-7.61 (m, 3H; 3 ', 4', 5'-Ar-H),
7.70-7.88 (m, 4H; 2,2 ', 5,5'-Ar-H).
¹³ C-NMR (CDCl ₃ ): δ (ppm)
50.6, 67.5, 69.5, 70.0, 70.6, 70.8, 113.9, 127.9,
129.5, 130.0, 131.6, 132.2, 138.0, 162.2, 195.1.
MALDI-TOF-MS m / z
Calculated value: 394.29,
Found: [M + K ⁺ ] 394.12.
Calculated value: 378.32.
Found: [M + Na ⁺ ] 378.14,
Calculated value: 356.35,
Found: [M + H ⁺ ] 356.16.

(2) Production of Compound 4 1.45 g (4.08 mmol) of Compound 3 and 100 mg of palladium-carbon (Pd: 10%) were mixed and suspended in 10 ml of ethanol. The mixture was stirred overnight at room temperature under hydrogen, and then diluted with chloroform. The solid was removed by Celite filtration, and the solvent was removed under reduced pressure to obtain Compound 4 as a yellow solid (1.24 g; yield 93%).
¹ H-NMR (CDCl ₃ ): δ (ppm)
3.15 (t, 2H; CH ₂ NH ₂ ), 3.55-3.97 (m, 8H; OCH ₂ ),
4.22 (t, 2H; Ar- OCH 2), 5.39 (br, 2H; NH 2),
7.00 (d, 2H; 3,5-Ar-H), 7.41-7.61 (m, 3H; 3 ', 4', 5'-Ar-H),
7.70-7.88 (m, 4H; 2,2 ', 5,5'-Ar-H).
¹³ C-NMR (CDCl ₃ ): δ (ppm)
39.8, 67.5, 69.4, 70.0, 70.6, 70.8, 114.1, 128.1,
129.6, 130.3, 131.9, 132.4, 138.0, 162.0, 195.5.
MALDI-TOF-MS m / z
Calculated value: 370.46
Found: [M + K ⁺ ] 368.13,
Calculated value: 352.41,
Found: [M + Na ⁺ ] 352.15
Calculated value: 330.43
Found: [M + H ⁺ ] 330.17.

(3) Production of Compound 6 500 mg (0.779 mmol) of known triazide dendron (Compound 5) and 254 mg (0.771 mmol) of Compound 4 and 200 μl of diisopropylethylamine (DIPEA) were mixed, and acetonitrile / chloroform (1/1 ) Dissolved in 10 ml. After adding 192 mg (0.818 mmol) of 1- [bis (dimethylamino) methylene] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), the mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate, washed successively with 2M aqueous sodium hydrogen sulfate solution, saturated brine, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. After filtration through alumina and removal of the solvent, purification was performed by column chromatography (silica gel, ethyl acetate / hexane = 4/1 → 1/0). This was further purified by recycled GPC to obtain Compound 6 as a pale yellow oily liquid (373 mg; yield 51%).
¹ H-NMR (CDCl ₃ ): δ (ppm)
3.36 (m, 6H; CH ₂ N ₃ ), 3.52-3.94 (m, 34H; OCH ₂ , NHCH ₂ ),
4.18 (m, 8H; Ar- OCH 2), 6.84 (t, 1H; CONHCH 2),
6.93 (d, 2H; 3,5-benzophenone-H), 7.08 (s, 2H; Ar-H),
7.40-7.60 (m, 3H; 3 ', 4', 5'-benzophenone-H),
7.67-7.83 (m, 4H; 2,2 ', 5,5'-benzophenone-H).
¹³ C-NMR (CDCl ₃ ): δ (ppm)
39.8, 50.6, 67.5, 69.0, 69.4, 69.7, 69.8, 69.9, 70.1, 70.4,
70.6, 70.7, 72.3, 106.9, 113.9, 128.0, 129.6, 130.1, 131.7,
132.3, 137.9, 141.2, 152.2, 162.0, 166.7, 195.5.
MALDI-TOF-MS m / z
Calculated value: 991.81,
Actual value: [M + K ⁺ ] 991.39,
Calculated value: 975.84,
Found: [M + Na ⁺ ] 975.42.

(4) Production of Compound 8 1.13 g (0.876 mmol) of known dendron (Compound 7) and 103 mg (1.87 mmol) of propargylamine were mixed and dissolved in 10 ml of acetonitrile. After adding 215 mg (0.920 mmol) of 1- [bis (dimethylamino) methylene] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), the mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate, washed successively with 2M aqueous sodium hydrogen sulfate solution, saturated brine, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. This was filtered through alumina and dried under reduced pressure to obtain Compound 8 as a white solid (661 mg; yield 57%).
¹ H-NMR (CDCl ₃ ): δ (ppm)
1.46 (m, 57H; CH ₃ ), 3.48-3.86 (m, 31H; OCH ₂ , CH ₂ -guanidine, CCH),
4.19 (m, 8H; Ar- OCH 2, CONHCH 2), 7.12 (br, 1H; CONHCH 2),
7.25 (s, 2H; Ar- H), 8.58 (br, 3H; CH 2 NHC (NBoc) (NHBoc)),
11.4 (br, 3H; NHBoc).
¹³ C-NMR (CDCl ₃ ): δ (ppm)
27.8, 28.0, 29.2, 40.3, 60.0, 68.6, 68.9, 69.5, 70.0,
70.1, 70.4, 70.8, 71.9, 78.8, 79.6, 82.5, 82.6, 106.8,
128.4, 140.9, 151.9, 152.4, 155.8, 162.9, 166.1.
MALDI-TOF-MS m / z
Calculated value: 1350.10,
Found: [M + Na ⁺ ] 1349.71,
Calculated value: 1274.48,
^{_{Found: [M-C 4 H 9}} + H +] 1271.66,
Calculated value: 1229.57,
Actual value: [M-Boc + H ⁺ ] 1227.67,
Calculated value: 1177.36,
Actual value: [M-Boc-C ₄ H ₉ + H ⁺ ] 1171.61,
Calculated value: 11128.38,
Found: [M-2Boc + H ⁺ ] 1127.62.
Calculated value: 1064.49,
^{_{Found: [M-2Boc-C 4}} H 9 + H +] 1071.56,
Calculated value: 1031.08,
Actual value: [M-3Boc + H ⁺ ] 1027.57,
Calculated value: 828.73,
Found: [M-5Boc + H ⁺ ] 827.46,
Calculated value: 727.22,
Actual value: [M-6Boc + H ⁺ ] 727.41.

(5) Preparation of compound 9 119 mg (0.125 mmol) of compound 6, 555 mg (0.418 mmol) of compound 8, copper sulfate pentahydrate 11 mg (0.04 mmol) and sodium ascorbate 166 mg (0.836 mmol) They were mixed, dissolved in 10 ml of tetrahydrofuran (THF) / water (1/1), and stirred at room temperature for 12 hours. The mixture was diluted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The solid was removed by Celite filtration, the solvent was removed under reduced pressure, and then purified by recycle GPC to obtain Compound 9 as a light brown solid (420 mg; yield 68%).
¹ H-NMR (CDCl ₃ ; ppm): δ
1.39 (m, 162H; CH ₃ ), 3.33-3.85 (m, 122H; OCH ₂ , CH ₂ -guanidine),
3.93-4.18 (m, 26H; Ar- OCH 2), 4.38 (br, 6H; CH 2 CH 2 -triazol),
4.53 (br, 6H; triazol-CH ₂ -NHCO), 6.82 (m, 3H; triazol-H),
7.08 (br, 6H; 3,5-benzophenone-H, CONH),
7.29 (br, 10H; Ar-H, 3,5-benzophenone-H),
7.33-7.53 (m, 3H; 3 ', 4', 5'-benzophenone-H),
7.55-7.86 (m, 4H; 2,2 ', 5,5'-benzophenone-H),
8.52 (br, 9H; CH 2 NHC (NBoc) (NHBoc)), 11.4 (br, 9H; NHBoc).
¹³ C-NMR (CDCl ₃ ; ppm): δ
27.9, 28.1, 35.2, 40.4, 49.9, 68.8, 69.0, 69.2, 69.5, 69.9,
70.2, 70.4, 70.5, 72.1, 78.8, 82.6, 106.9, 113.8, 123.2, 127.8,
128.8, 129.3, 129.5, 129.9, 131.5, 132.0, 137.8, 141.1, 152.1,
152.6, 155.8, 161.9, 163.0, 166.4, 166.6, 194.8.

(6) Production of G1-BP 405 mg (0.082 mmol) of Compound 9 was dissolved in 5 ml of 4.0 M hydrogen chloride dioxane solution, stirred at room temperature for 6 hours, and then dried under reduced pressure to give G1-BP a light brown As a solid (282 mg; yield> 99%)
¹ H-NMR (D ₂ O; ppm): δ
3.28 (m, 18H; CH ₂ -guanidine),
_{3.37-4.23 (m, 106H; OCH 2} , CH 2 CH 2 NHCO),
4.36 (m, 26H; Ar- OCH 2),
_{4.59 (br, 12H; CH 2} CH 2 -triazol, triazol-CH 2 -NHC),
6.65 (m, 3H; triazol-H), 6.94-7.15 (m, 10H; Ar-H, 3,5-benzophenone-H),
7.19-7.56 (m, 7H; 3 ', 4', 5'-benzophenone-H, 2,2 ', 5,5'-benzophenone-H).
¹³ C-NMR (D ₂ O; ppm): δ
29.0, 40.52, 42.0, 51.4, 67.2, 69.0, 69.5, 69.6, 69.7, 70.1, 70.3, 70.4,
70.7, 72.7, 106.6, 106.8, 114.7, 129.6, 130.2, 130.3, 133.2, 133.4,
137.4, 140.2, 152.4, 152.6, 157.8, 162.9, 166.7, 167.5, 168.8, 198.0.

Production of G1-NBP The objective dendrimer G1-NBP of the present invention was produced according to the following chemical reaction formula.

(1) Preparation of Compound 11 330 mg (1.17 mmol) of known triethylene glycol derivative (Compound 10), 500 mg (0.779 mmol) of Compound 5, diisopropylethylamine with amino group protected with benzyloxycarbonyl group (Cbz) (DIPEA) 171 μl was mixed and dissolved in 6 ml of acetonitrile. After adding 190 mg (0.810 mmol) of 1- [bis (dimethylamino) methylene] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), the mixture was stirred at room temperature for 1 hour. After removing the solvent under reduced pressure, the residue was purified by column chromatography twice (alumina, ethyl acetate + 10% methanol) and (silica gel, ethyl acetate) to obtain Compound 11 as a light yellow oily liquid (469 mg; yield 66). %).
¹ H-NMR (CDCl ₃ ; ppm): δ
3.36 (m, 8H; CH ₂ N ₃ , CH ₂ NHCO ₂ Bn),
_{3.51-3.88 (m, 34H; OCH 2} , CH 2 NHCO), 4.18 (m, 6H; Ar-OCH 2),
5.06 (s, 2H; PhCH 2 O), 7.04 (s, 2H; Ar-H), 7.31 (br, 5H; Ph-H).
¹³ C-NMR (CDCl ₃ ; ppm): δ
50.6, 61.7, 66.7, 69.0, 69.7, 69.9, 70.1, 70.4, 70.6, 70.7, 72.3,
107.0, 127.9, 128.3, 129.6, 136.3, 141.1, 152.2, 156.2, 166.8.
MALDI-TOF-MS m / z
Calculated value: 944.17,
Found: [M + K ⁺ ] 944.39,
Calculated value: 928.19,
Found: [M + Na ⁺ ] 928.41.

(2) Production of Compound 12 410 mg (0.453 mmol) of Compound 11 was dissolved in 4 ml of THF and cooled to 0 ° C. After adding 392 mg (1.49 mmol) of triphenylphosphine, the temperature was gradually raised to room temperature and then stirred at room temperature for 3 hours. After adding 1 ml of water, the mixture was further stirred at 50 ° C. overnight. After returning to room temperature, 3 ml of water and 450 μl of 12M hydrochloric acid were added, followed by washing with toluene three times. The solvent was removed under reduced pressure to obtain Compound 12 as a brown oily liquid (423 mg; yield> 99%).
¹ H-NMR (D ₂ O; ppm): δ
3.08 (m, 8H; CH ₂ N ₃ , CH ₂ NHCO ₂ Bn),
3.35-3.88 (m, 34H; OCH ₂ , CH ₂ NHCO),
4.03-4.20 (m, 6H; Ar- OCH 2), 4.84 (s, 2H; PhCH 2 O),
7.00 (s, 2H; Ar-H), 7.11-7.27 (m, 5H; Ph-H).
¹³ C-NMR (D ₂ O; ppm): δ
40.2, 61.4, 67.4, 69.3, 69.9, 70.1, 70.5, 70.8, 71.0, 73.1,
107.5, 128.3, 129.0, 129.5, 130.4, 137.3, 140.5, 152.6, 158.7, 169.7.
MALDI-TOF-MS m / z
Calculated value: 866.00,
Found: [M + K ⁺ ] 866.42,
Calculated value: 850.04
Actual value: [M + Na ⁺ ] 850.44
Calculated value: 828.07,
Found: [M + H ⁺ ] 828.46.

(3) Production of Compound 13 1.71 g (1.33 mmol) of Compound 7, 377 mg (0.402 mmol) of Compound 12 and 292 μl of diisopropylethylamine (DIPEA) were mixed and dissolved in 10 ml of acetonitrile. After adding 324 mg (1.38 mmol) of 1- [bis (dimethylamino) methylene] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), the mixture was stirred at room temperature for 4.5 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate and filtered through alumina. Again, the solvent was removed under reduced pressure, dissolved in chloroform, and reprecipitated in hexane to obtain Compound 13 as a pale yellow solid (1.13 g; yield 60%).
¹ H-NMR (CDCl ₃ ; ppm): δ
1.45 (m, 162H; CH ₃ ),
3.42-3.86 (m, 132H; OCH ₂ , CH ₂ -guanidine, CH ₂ NHCO),
3.86 (m, 24H; Ar- OCH 2), 7.07 (br, 8H; Ar-H), 7.22 (m, 5H; Ph-H),
8.56 (br, 9H; CH 2 NHC (NBoc) (NHBoc)), 11.4 (br, 9H; NHBoc).
¹³ C-NMR (CDCl ₃ ; ppm): δ
28.0, 28.3, 38.5, 39.8, 40.5, 43.4, 66.4, 68.9, 69.2, 69.6, 70.1,
70.3, 70.5, 72.2, 79.1, 82.8, 106.9, 127.7, 127.8, 128.2,
129.4, 129.5, 141.1, 152.2, 152.6, 155.9, 163.1, 166.8.

(4) Production of Compound 14 931 mg (0.200 mmol) of Compound 13 and 50 mg of palladium-carbon (Pd: 10%) were mixed and suspended in 10 ml of ethanol. The mixture was stirred overnight at room temperature under hydrogen, and then diluted with chloroform. The solid was removed by Celite filtration, and the solvent was removed under reduced pressure to obtain a white solid (852 mg; yield 94%).
¹ H-NMR (CDCl ₃ ; ppm): δ
1.41 (m, 162H; CH ₃ ), 3.07 (br, 2H; CH ₂ NH ₂ ),
3.38-3.93 (m, 130H; OCH ₂ , CH ₂ -guanidine, CH ₂ NHCO),
4.11 (m, 24H; Ar- OCH 2), 7.09 (m, 8H; Ar-H),
8.27 (br, 2H; NH ₂ ), 8.56 (br, 9H; CH ₂ NHC (NBoc) (NHBoc)),
11.2 (br, 9H; NHBoc).
¹³ C-NMR (CDCl ₃ ; ppm): δ
27.2, 27.4, 39.0, 41.3, 42.1, 53.8, 68.1, 68.5, 68.8, 68.9,
69.6, 69.8, 71.6, 81.6, 84.2, 106.1, 126.9, 127.1, 127.6,
128.8, 135.9, 139.7, 151.3, 154.4, 155.8, 157.9, 166.2.

(5) Preparation of Compound 15 476 mg (0.106 mmol) of Compound 14, 638 mg (2.67 mmol) of known benzophenone-4-isothiocyanate and 368 μl of triethylamine were mixed, dissolved in 5 ml of DMF, and stirred at room temperature overnight. . The mixture was diluted with ethyl acetate, washed successively with saturated brine, saturated aqueous ammonium chloride and saturated brine, and dried over anhydrous sodium sulfate. Purification by two reprecipitations (chloroform → hexane, chloroform → diethyl ether) gave compound 15 as a brown solid (375 mg; yield 75%).
¹ H-NMR (CDCl ₃ ; ppm): δ
1.44 (m, 162H; CH ₃ ), 3.34-3.84 (m, 132H; OCH ₂ , CH ₂ -guanidine,
_{CH 2 NHCO, CH 2 NHCS)} , 4.22 (m, 24H; Ar-OCH 2),
7.06 (m, 8H; Ar-H), 7.32-7.99 (m, 9H; benzophenone-H),
8.57 (br, 9H; CH 2 NHC (NBoc) (NHBoc)), 11.4 (br, 9H; NHBoc).
¹³ C-NMR (CDCl ₃ ; ppm): δ
28.1, 28.3, 39.9, 40.6, 68.5, 68.9, 69.3, 69.8, 70.3, 70.4,
72.5, 79.2, 83.0, 102.8, 107.0, 117.7, 125.3, 128.1, 128.2,
128.3, 129.7, 129.9, 131.5, 131.8, 138.0, 140.0, 152.3,
152.7, 156.3, 162.1, 163.5, 195.5.

(6) Production of G1-NBP 325 mg (0.068 mmol) of Compound 15 was dissolved in 5 ml of 4.0 M hydrogen chloride dioxane solution, stirred at room temperature for 8.5 hours, and then dried under reduced pressure. This was washed with chloroform to obtain G1-NBP as a dark brown solid (151 mg; yield 67%).
¹ H-NMR (d ₆ DMSO; ppm): δ
2.85-3.15 (m, 18H; CH ₂ -guanidine),
3.17-3.81 (m, 108H; OCH ₂ , CH ₂ CH ₂ NHCO),
3.90-4.24 (m, 24H; Ar- OCH 2),
6.92-7.20 (m, 10H; Ar-H, 3,5-benzophenone-H),
7.32-7.85 (m, 7H; 3 ', 4', 5'-benzophenone-H, 2,2 ', 5,5'-benzophenone-H).
¹³ C-NMR (D ₂ O; ppm): δ
40.6, 42.3, 69.4, 69.9, 70.9, 73.2, 107.6, 119.1, 129.2, 130.6,
131.6, 132.2, 133.3, 138.0, 141.1, 152.7, 158.2, 168.6, 196.6.

Protein intracellular uptake (1) Adjustment of labeled protein As a target protein, fluorescently labeled actin was prepared by the following method.
1 mg of actin (from rabbit skeletal muscle) was dissolved in 500 μl of 20 mM HEPES buffer (containing 25 mM potassium chloride, pH 7.0). To this, 50 μl of Alexa Fluor (registered trademark) 633 C ₅ -maleimide (1 mM) HEPES buffer solution was added and allowed to stand at room temperature for 4 hours. After removing the excess fluorescent dye with the desalting column G-25, it was diluted with HEPES buffer to 200 μg / ml, dispensed, and stored at −20 ° C.
(2) Evaluation of protein uptake activity Evaluation of protein uptake activity was performed by the following method.
HeLa cells were cultured in a 6-well culture dish at 100,000 cells / well in a DMEM medium containing calf serum (FBS; 10%). Various reagents shown below and Alexa Fluor (registered trademark) 633-labeled actin were mixed, and then diluted with the DMEM medium containing no FBS to the concentrations shown below. This was added to HeLa cells washed with PBS, and contacted at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. The HeLa cells were washed again with PBS three times, and the cells detached from the dish with trypsin-EDTA were analyzed by flow cytometry, and the fluorescence intensity of Alexa Fluor (registered trademark) 633 bound to actin was measured. For comparison, the case where only fluorescently labeled actin was contacted was measured. The result shown in FIG. 1 was obtained.

Concentrations at the time of contact of the used proteins and reagents are as follows.
(1) Fluorescently labeled actin: 50 nM (common)
(2) Fluorescently labeled actin + arginine nonamer (Arg ₉ ): 1 μM
(3) Fluorescently labeled actin + G1-NOMe: 1 μM
(4) Fluorescently labeled actin + G1-NFITC: 1 μM
(5) Fluorescently labeled actin + SAINT-phD: 15 μM
(6) Fluorescently labeled actin + G1-BP: 1 μM
In addition, the arginine nonamer (Arg ₉ ) of (2) used as a comparative example is a cell membrane permeable peptide, and G1-NOMe of (3) is represented by the following chemical formula:

A polyion dendrimer having no benzophenone group in the core represented by the formula (4), wherein G1-NFITC is represented by the following chemical formula:

Is a polyion dendrimer having a 9-phenylxanthene derivative instead of a benzophenone group at the core, and SAINT-phD in (5) is a commercially available protein introduction agent.
As shown in FIG. 1, Arg ₉ known as a membrane permeable peptide showed almost no difference from the case where Arctin alone was contacted. When SAINT-phD, which is a commercially available protein introduction agent, was used, significantly higher uptake activity was seen compared to Arg ₉ .
On the other hand, when the G1-BP of the present invention was used, it showed a three-fold or higher uptake activity even though the concentration was 1/15 of SAINT-phD.
In addition, G1-NOMe and G1-NFITC, which are polyion dendrimers having no benzophenone group in the core part, showed almost no difference from the case where only actin was contacted.
As a result, it was confirmed that the polyion dendrimer in which a substituent having a benzophenone group is bonded to the core of the present invention has a remarkable protein transporting action compared to other polyion dendrimers.

Confirmation of protein uptake by observation with a confocal microscope Further, the uptake of Alexa Fluor (registered trademark) 633-labeled actin by G1-BP of the present invention into cells was confirmed as follows.
HeLa cells were cultured at 10,000 cells / plate in a glass bottom culture dish for microscopic observation. G1-BP and Alexa Fluor (registered trademark) 633-labeled actin were mixed and then diluted with DMEM medium without FBS. This was added to HeLa cells washed with PBS, and contacted at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. After washing 3 times with PBS, DMEM medium was added and observed with a confocal microscope (excitation wavelength: 633 nm) to obtain the results of FIG.
Concentrations at the time of contact of the used proteins and reagents are as follows.

(1) Fluorescently labeled actin: 50 nM
(2) Fluorescently labeled actin + G1-BP: 1 μM

The left side of FIG. 2 shows the case where G1-BP of the present invention is mixed, and the right side shows the case where only fluorescently labeled actin is used. As shown on the left side of FIG. 2, when the G1-BP of the present invention is mixed, fluorescence derived from Alexa Fluor (registered trademark) 633 is observed inside the cell, and the labeled actin is taken up into the HeLa cell. I was able to confirm.

Influence of serum In order to examine the influence of serum on protein uptake activity, a similar experiment was performed using a medium containing 10% FBS. The specific operation is as follows.
HeLa cells were cultured in a 6-well culture dish at 100,000 cells / well in a DMEM medium containing calf serum (FBS; 10%). Various reagents shown below and Alexa Fluor (registered trademark) 633 labeled actin were mixed, and then diluted with DMEM medium (10% FBS). This was added to HeLa cells washed with PBS, and contacted at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. HeLa cells were washed again with PBS three times, and the cells detached from the dish with trypsin-EDTA were analyzed by flow cytometry, and the results shown in FIG. 3 were obtained. The left side of FIG. 3 shows the case where SAINT-phD is mixed, and the right side shows the case where G1-BP of the present invention is mixed. The left side (blue) shows the case without serum, and the right side (yellow) shows the case with serum.

Concentrations at the time of contact of the used proteins and reagents are as follows.
Fluorescently labeled actin: 50 nM (common)
(1) Fluorescently labeled actin + SAINT-phD: 15 μM
(2) Fluorescently labeled actin + G1-BP: 1 μM

FIG. 3 shows the results of SAINT-phD and G1-BP without serum and with serum, respectively. The G1-BP of the present invention exhibits sufficiently high protein uptake activity even in the presence of serum, and still exhibits more than twice the activity of SAINT-phD.

Effect of Concentration and Contact Time In order to examine the influence of the contact concentration and contact time of G1-BP on the protein uptake amount, the following experiment was performed.
HeLa cells were cultured in a 6-well culture dish at 100,000 cells / well in a DMEM medium containing calf serum (FBS; 10%). Various reagents and Alexa Fluor (registered trademark) 633 labeled actin were mixed, and then diluted with DMEM medium not containing FBS. This is added to HeLa cells washed with PBS, and at 37 ° C., 5% CO ₂ atmosphere for 1 to 4 hours under the condition of G1-BP (1 μM), or G1-BP (0.05, 0.1, 0). .5, 1 μM) for 4 hours. The HeLa cells were washed again with PBS three times, and removed from the dish with trypsin-EDTA, and analyzed by flow cytometry, and the results shown in FIGS. 4 (a) and (b) were obtained. (A) [upper side] of FIG. 4 shows the results when contacted for 1 to 4 hours under the condition of G1-BP (1 μM) of the present invention, and FIG. The results are shown when the G1-BP is contacted at a concentration of 0.05 μM, 0.1 μM, 0.5 μM, or 1 μM for 4 hours.
When 1 μM of G1-BP of the present invention was used, performance exceeding the activity at the time of SAINT-phD contact for 4 hours was exhibited even if the contact time was 1 hour. In the SAINT-phD protocol, the incubation time for introduction into the cell is 4 hours, whereas when the polycationic dendrimer of the present invention is used at a concentration of 1 μM, a rapid protein that exceeds this in 1 hour. It became clear that transportation was achieved. In addition, in the case of contact for 4 hours, even when the concentration is as low as 0.5 μM (1/30 of SAINT-phD), it is 2.5 times that when a commercially available protein introduction reagent SAINT-phD (cationic lipid) is used at a concentration of 15 μM The above protein transport activity was shown.

Presence or absence of protein denaturation It was confirmed by the following experiment whether the protein taken up by G1-BP in the cell maintained its activity.
HeLa cells were cultured at 10,000 cells / plate in a glass bottom culture dish for microscopic observation. G1-BP and green fluorescent protein (GFP) were mixed and then diluted with DMEM medium without FBS. This was added to HeLa cells washed with PBS, and contacted at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. After washing 3 times with PBS, DMEM medium was added and observed with a confocal microscope (excitation wavelength 488 nm), and the results shown in FIG. 5 were obtained. The left side of FIG. 5 shows the result when G1-BP of the present invention is mixed, and the right side shows the result when only green fluorescent protein (GFP) is used.
Concentrations at the time of contact of the used proteins and reagents are as follows.

Green fluorescent protein (GFP): 5 nM
G1-BP: 1 μM

Since GFP-derived fluorescence incorporated into the cells can be confirmed, it can be seen that the incorporated protein could maintain its function without causing denaturation or inactivation.

Cytotoxicity The cytotoxicity of G1-BP was examined using Cell Counting Kit-8 (Dojindo Laboratories) as follows.
HeLa cells were cultured on a 96-well culture plate at 5,000 cells / well. 100 μl of DMEM medium (without FBS) containing G1-BP was added to these HeLa cells washed with PBS, and contacted at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. Here, 10 μL of Cell Counting Kit-8 coloring reagent was added, and after standing at 37 ° C. and 5% CO ₂ atmosphere for 1 hour, toxicity was evaluated by measuring absorbance at 450 nm, and the result shown in FIG. 6 was obtained. It was. The vertical axis of FIG. 6 indicates the cell viability (ratio), and the horizontal axis indicates the concentration of G1-BP (μM).
As shown in FIG. 6, no cytotoxicity occurs even at a concentration of 1 μM at which the protein uptake activity is sufficiently exerted. As described above, the G1-BP of the present invention did not show any toxicity to the target cells, and showed a higher protein uptake activity than the existing SAINT-phD.

  The present invention provides a novel polycationic dendrimer (dendritic high molecular weight) as a protein carrier molecule that can rapidly and efficiently transport a target protein into a cell without impairing the activity of the protein. Molecule) and is useful as an efficient and rapid protein transporter into cells.

Claims (10)

  The dendrimer has a cationic group selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group, a polyalkyleneoxy group as a branched chain, and a benzophenone group in the core portion. A polyion dendrimer, wherein a substituent is bonded.   2. The polyion dendrimer according to claim 1, wherein the branched chain is a polyethyleneoxy group having a number of repetitions of 1 to 6, which may have a 1,2,3-triazole group bonded to the terminal at the 1,4-position.   The polyion dendrimer according to claim 1 or 2, wherein the branched portion is composed of 3,4,5-trihydroxybenzoic acid amide or 3,4,5-trihydroxycinnamic acid amide. The substituent having a benzophenone group is represented by the following general formula [2] or [3],
[Wherein, k represents 0 or an integer of 1 to 5. ]
[Wherein, k represents 0 or an integer of 1 to 5. ]
The polyion dendrimer according to any one of claims 1 to 3, which is a substituent having a benzophenone group represented by the formula:   The polyion dendrimer according to any one of claims 1 to 4, wherein the generation is 1 to 5 generations. The polyion dendrimer has the following general formula [1]
[Wherein X represents the following general formula [2] or [3],
[Wherein, k represents 0 or an integer of 1 to 5. ]
[Wherein, k represents 0 or an integer of 1 to 5. ]
And Y represents a cationic group independently selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group, and l represents 1-6. Represents an integer, m represents 0 or 1, n represents the number of generations, and represents an integer of 1 to 5; The branched 3,4,5-trihydroxybenzoic acid amide in the formula may be 3,4,5-trihydroxycinnamic acid amide, and the branched ethyleneoxy chain has 1 to 1 carbon atoms. It may be 6, preferably 2-5 linear or branched alkylene chains. ]
The polyion dendrimer according to any one of claims 1 to 5, which is represented by the following formula. The polyion dendrimer has the following general formula [4]
[Wherein Y represents a cationic group independently selected from the group consisting of a guanidine group, a thiourenium group, and an isothiourenium group, and m represents 0 or 1. ]
The polyion dendrimer according to any one of claims 1 to 6, wherein The polyion dendrimer has the following formula [5]
The polyion dendrimer according to any one of claims 1 to 7, which is represented by the following formula.   A protein transporter comprising the polyion dendrimer according to any one of claims 1 to 8.   A method for transporting a protein, comprising using the protein transport agent according to claim 9.