JP2003261574A - Biotin lipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biotin lipid having a new structure imparting a function fixing a protein usable for cell culture on the surface of a material by coating the biotin lipid on a hydrophobic surface. <P>SOLUTION: This biotin lipid is represented by formula (1); (wherein, R<SB>1</SB>is O(CH<SB>2</SB>)<SB>n</SB>CH<SB>3</SB>; R<SB>2</SB>is H or R<SB>1</SB>; m is an integer of 2-6; and n is an integer of 11-17.). The compound (biotin lipid) fixing the protein on several forms of the culture materials is synthesized from a commercially available material at a high yield in a short process. The biotin lipid can be coated on the hydrophobic surface and avidin can be fixed by using the biotin with an intermolecular interaction analyzer. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biotin lipid having a novel structure. More specifically, the present invention relates to a biotin lipid having a novel structure which has a function of fixing a proteinaceous factor used for cell culture or the like on the surface of a material by coating the surface with hydrophobic.

[0002]

2. Description of the Related Art Utilization of stem cells and somatic cells has been actively studied in the fields of regenerative medicine and artificial organs. Various adhesion factors for adhesion, proliferation, differentiation, activation, etc. of such cells,
Protein factors such as growth factors, differentiation inducers and cytokines are used. Since such proteinaceous factors are generally expensive and inefficiently used as a component of a culture solution, attempts have been made to immobilize them on a culture material and significantly reduce the amount used.

Proteins involved in cell adhesion include fibronectin, laminin, tenascin, vitronectin and the like, and it has already been found that cell adhesion can be enhanced by immobilizing or coating these on artificial materials. Known (Y. Ikada et al., Biomateri
als. , 12, 747-751, 1991).
Therefore, it has been studied to immobilize proteinaceous factors on the materials used for cell culture, but due to problems such as protein denaturation, the method depends on the culture materials and proteins used. However, there is no general method.

On the other hand, the binding method between proteins utilizing the strong interaction of biotin-avidin is widely used as a modification method while keeping the activity of the protein (Neochemistry Chemistry Laboratory, Volume 12, Molecular Immunology). III Antigen / Antibody / Complement, pp. 99-104, edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1992)

As a study of immobilizing a protein on the surface of a material using biotin-avidin, for example, in the case of using a polymer, avidin and polyvinylsulfonic acid are alternately adsorbed on a silicon-coated glass substrate. There is a study in which avidin was immobilized by allowing the binding and biotin binding to the avidin was confirmed. Various functional molecules labeled with biotin can be immobilized using this immobilized avidin (J. Anzai et al., Che.
m. Soc. Perkin Trans. 2,2413
P., 1999).

In the case of a low molecular weight compound, it has been reported that a biotin lipid using alkanethiol as a lipid moiety is bound to a gold surface to immobilize a protein via avidin. Ringsdorf et al. Immobilize biotin lipids in which alkanethiol is bound to biotin on a gold surface, and each molecule recognizes streptavidin and biotin-labeled anti-HCG (human chorion).
It has been confirmed by a surface plasmon resonance device that the ic gonadotropin) -Fab and the Fab fragment are sequentially bound. By this method, it is shown that the protein can be immobilized by using biotin immobilized on the surface of the substrate, and that the immobilized protein is functioning (H. Ringsdorf.
Et al., Science, Volume 262, 1706-1708,
1993).

Similarly, as an example of a biotin lipid having an alkanethiol in the lipid moiety, the interaction between biotin lipid immobilized on a gold surface and streptavidin is measured, and biotin-labeled bovine serum albumin is bound to the avidin. There is an example of trying (H. J. Gruber et al.,
Tetrahedron Letters, Volume 42, 2
677-2680, 2001). This method also shows that protein can be immobilized by using immobilized biotin.

[0008] However, since these methods use alkanethiol, they can be used only for a special material such as a gold surface, and are therefore a general method for immobilizing a protein on a culture material. I don't get it.

[0009]

The object of the present invention is to provide a novel structure having a function of fixing a proteinaceous factor used for cell culture or the like on the material surface by coating the hydrophobic surface. To provide biotin lipids.

[0010]

[Means for Solving the Problems] The present inventors can immobilize proteins on culture materials of various shapes by a simple operation that can be synthesized from commercially available inexpensive raw materials in a short process at high yield and by coating. It was confirmed that the biotin lipid can be coated on the hydrophobic surface and that avidin can be immobilized by using the biotin, by synthesizing a compound to be converted, and using an intermolecular interaction analyzer.

[Chemical 2] (In the formula, R ₁ represents an O (CH ₂ ) _n CH ₃ group, R ₂ represents H or R ₁ , m is an integer of 2 to 6, and n is 11 to 17
Represents the integer. )

The method for synthesizing the compound of the present invention may be any method. A benzoic acid derivative having a long-chain alkyloxy group bonded at the 3, 4, and 5 positions can be obtained from commercially available compounds by a method known from the literature (VSK Balagurusami et al.
J. Am. Chem. Soc. 119, 1539-
1555, 1997). Also 3, 5
Similarly, a benzoic acid derivative having a long-chain alkyloxy group bonded at the position may be prepared by a method known in the literature (V. Percec et al., Angew.
Chem. Int. Ed. , 39, 1598-160
Page 2, 2000).

The benzoic acid derivative thus obtained is hydrolyzed to give a free carboxylic acid, which is condensed with a compound in which an alkanediamine or one of the amines is protected according to a method generally used for amide bond formation. By deprotecting according to the above, a lipid in which benzoic acid is bound to one amine of alkanediamine can be obtained. A general amide condensation reaction can be used to bond the amine compound thus obtained with biotin.

To immobilize a protein, the synthesized biotin lipid is dissolved in a suitable solvent such as ethanol and coated on a hydrophobic surface such as a cell culture material, and then a solution of avidin-labeled protein is added. The protein can be immobilized by immobilizing the protein by once or by immobilizing avidin and then adding a biotin-labeled protein solution. The modification with avidin or biotin has been established as a method for modifying while maintaining the activity of the protein (Shinsei Chemistry Laboratory, Volume 12,
Molecular Immunology III Antigen / Antibody / Complement, pp. 99-104,
This method can be used as a general method for protein immobilization, as described in The Biochemical Society of Japan, Tokyo Kagaku Dojin, 1992).

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a biotin lipid represented by the formula (1), and a protein immobilizing material containing the biotin lipid represented by the formula (1) as an active ingredient. The present invention provides a protein immobilizing agent containing a biotin lipid represented by the formula (1) as an active ingredient.

The present invention will be described in more detail below, but the present invention is not limited to the following description.

[0016]

【Example】

Example 1 N- (N-biotinyl-6-aminohexyl)
Synthesis of -3,5-bis (dodecyloxy) benzamide (+)-biotin (245 mg, 1.00 mmol), triphenylphosphine (394 mg, 1.50 mmol), triethylamine
(100 μl, 1.00 mmol), carbon tetrachloride (230 μl, 1.50 mmol)
Was dissolved in dichloromethane and N- (6-aminohexyl) -3,5-bis (dodecyloxy) benzamide (470 m
g, 0.08mmmol) in dichloromethane at room temperature.
Stir for hours. After completion of the reaction, the reaction solution was concentrated and purified by silica gel column chromatography (chloroform: methanol = 96: 4) to obtain the target compound (390 mg, 0.48 m
mol, 60%) was obtained.

1 H NMR (CDCl ₃ ) δ 0.88 (6H, t, J = 6.8 H
z), 1.25-1.80 (52H, m), 2.20 (2H, m), 2.71 (1H, d,
J = 12.9 Hz), 2.90 (1H, dd, J = 12.9, 5.1 Hz), 3.14 (1
H, td, J = 7.3, 5.1 Hz), 3.23 (2H, q, J = 6.6 Hz), 3.4
1 (2H, q, J = 6.6 Hz), 3.96 (4H, t, J = 6.6 Hz), 4.31
(1H, dd, J = 7.6, 5.1 Hz), 4.49 (1H, dd, J = 7.6, 5.1H
z), 4.31 (1H, dd, J = 7.6, 5.1 Hz), 5.00 (1H, s), 5.
83 (1H, s), 6.00 (1H, t, J = 5.1 Hz), 6.49 (1H, t, J
= 5.1 Hz), 6.55 (1H, t, J = 2.2 Hz), 6.90 (2H, d, J =
2.2 Hz).

[0018]

Example 2 Interaction between biotin lipid and streptavidin The interaction between biotin lipid (1) obtained above and streptavidin is IAs which is an intermolecular interaction analyzer.
The analysis was performed using ys Plus (manufactured by Affinity Sensors).

The hydrophobic cuvette was washed with a surfactant, a buffer and 2-propanol, and then N- (N-biotinyl-6-aminohexyl) -3,5-bis (dodecyloxy).
Immobilization was carried out by adding a 0.8 μM solution of benzamide (2-propanol: chloroform = 19: 1). The immobilized amount was 755 Arc seconds.

The cuvette was washed with a buffer, an aqueous solution of hydrochloric acid and an aqueous solution of sodium hydroxide, blocked with bovine serum albumin, washed with a buffer, and added with 50 μl of streptavidin 40 μg / ml,
The interaction was examined.

The adsorption amount after 5 minutes was 620 Arc sec.
and adsorption amount after 10 minutes 620 Arcs
It was confirmed that econds and streptavidin were hardly dissociated while being immobilized.

[0022]

INDUSTRIAL APPLICABILITY The present invention provides a biotin lipid having a novel structure, which is coated on a hydrophobic surface to impart a function of immobilizing a proteinaceous factor used in cell culture or the like on the material surface. Is.

Claims (3)

[Claims] 1. A biotin lipid represented by the formula (1). [Chemical 1] (In the formula, R ₁ represents an O (CH ₂ ) _n CH ₃ group, R ₂ represents H or R ₁ , m is an integer of 2 to 6, and n is 11 to 17
Represents the integer. ) 2. A protein immobilizing material containing a biotin lipid represented by the formula (1) as an active ingredient. 3. A protein immobilizing agent containing a biotin lipid represented by the formula (1) as an active ingredient.