JPWO2016158327A1 - Compound for immobilizing lipid membrane-containing material, substrate modified with the compound, method for patterning lipid membrane-containing material on the substrate, and method for isolating lipid membrane-containing material on the substrate - Google Patents

Abstract

The present invention relates to a compound of the formula (I) wherein A1 is a lipid membrane-binding group that binds to a lipid membrane in a lipid membrane-containing product, and A2 represents the binding of the lipid membrane-binding group to the lipid membrane. A group that inhibits lipid membrane binding, A3 is a photoreactive group, A4 is a hydrophilic group, A5 is a reactive group to the substrate to be modified, and A6 is at least three bonds A scaffold site having a hand, L1 and L2 are linkers, and k1 and k2 are each independently 0 or 1, and a lipid membrane binding inhibitory group by photoreaction Inhibition of binding by the above-mentioned compounds, and the lipid membrane binding group can bind to the lipid membrane, the compound, a substrate modified with the compound, a method of patterning a lipid membrane-containing material on the substrate, and the The present invention relates to a method for isolating a lipid membrane-containing material on a substrate.

Description

  The present invention relates to a compound for immobilizing a lipid membrane-containing material, a substrate modified with the compound, a method for patterning a lipid membrane-containing material on the substrate, and a lipid membrane-containing material on the substrate. Relates to the method of releasing.

  In tissues in a living body, a plurality of types of cells are arranged at specific positions and organized to express normal functions. Development of a technique for arranging a plurality of types of cells at arbitrary positions that enables the construction of a tissue for transplantation or a tissue model that expresses a function similar to that of a tissue in a living body is required.

  In addition, by using a substrate on which many types of cells are arranged, drug screening for many types of cell samples can be performed at once. Therefore, it is possible to increase the throughput of the assay and save the reagent by the technique of arranging a plurality of types of cells at arbitrary positions.

  As a technique that makes it possible to produce a pattern composed of a plurality of types of cells, a method using a plurality of types of cells with high cell specificity is known (Non-Patent Document 1).

  In addition, a method using a functional polymer that enables cells to adhere spontaneously by light irradiation is known (Non-Patent Documents 2 and 3).

  Furthermore, a technique is known in which complementary DNA is modified on a cell and a base material, and a pattern composed of a plurality of types of cells is produced using their complementarity (Non-patent Document 4).

  Further, BAM (Biocompatible Anchor for cell Membrane) is known as a biocompatible anchor capable of modifying the cell surface without causing damage to the cells (Non-patent Document 5). BAM is an amphiphilic compound having an oleyl group which is a hydrophobic aliphatic hydrocarbon group and a PEG group which is a hydrophilic group. It is possible to modify the cell membrane by the non-covalent bond between the hydrophobic site of BAM and the cell membrane which is a lipid membrane. It has also been reported that cells and liposomes can be immobilized by binding a carrier (including BAM) having a hydrophobic chain and a hydrophilic chain to a substrate (Patent Document 1).

  Cancer is also known to cause distant metastasis. Some cancer cells in the primary lesion enter the blood vessel, circulate in the blood as circulating cancer cells (CTC) in the blood, and form a metastatic lesion at a site away from the primary cancer. Many causes of cancer patient death are distant metastases. Therefore, early detection of CTCs from blood samples of cancer patients can determine the presence of distant metastases and the effects of surgery, radiation therapy, molecular targeted drug therapy, or chemotherapy on the primary cancer, It becomes possible to contribute to prolonging the improvement of the survival rate of cancer patients. Furthermore, it is expected that CTC can be analyzed by isolating and culturing CTC while alive.

  The number of CTCs is very small compared to the number of white blood cells contained in blood. The number of CTCs is about 10 for 5 billion red blood cells and 3 million to 10 million white blood cells per mL of blood. Therefore, in order to detect CTC, only a small number of cancer cells are concentrated using some method from a group of cells in which a large number of blood cells and a small number of cancer cells are mixed, and further, blood cells And cancer cells must be identified using some method.

International Publication No. 2003/074691

Lab Chip, 2010, 10, 3413-3421, 3413 Langmuir 2008, 24, 13084-13095 Biotechnology and Bioengineering, Vol. 103, No. 3, June 15, 2009, 552-561 Angew. Chem. Int. Ed. 2011, 50, 7378 -7380 Bio Techniques, 2003, Vol. 35, No. 5, 1014-1021

  As in Non-Patent Document 1, a method using a plurality of types of antibodies with high cell specificity must use expensive antibodies. In addition, since only specific cells are bound by the antibody, it is necessary to consider the combination of the antibody and the cell, and it is necessary to search for a complicated combination. In addition, since some cells have not obtained specific antibodies, they lack general versatility. Furthermore, since it is easily decomposed and denatured by mixing enzymes such as protease and denaturing agents, there is a problem that strict quality control is required.

  In addition, in the method using a functional polymer that allows cells to adhere spontaneously by light irradiation as in Non-Patent Documents 2 and 3, in order to utilize cell adhesion, non-adherent cells and weakly adherent cells can be used. It cannot be applied. Moreover, it takes several hours to one day or more to pattern one type of cell, and there is a problem that throughput is low.

  Further, in the method using the complementarity of DNA as in Non-Patent Document 4, it is necessary to modify the DNA on the cell surface in advance, so that the work becomes complicated. In addition, DNA is difficult to synthesize because it is difficult to synthesize at a mass scale of more than mg scale. Furthermore, since it is easily decomposed by contamination with an enzyme such as DNase, there is a problem that strict quality control is required.

  There is also a need for a technique that can specifically isolate CTCs that are contained in blood in very small amounts. There is also a need for a technique that can isolate a specific lipid membrane-containing material from a mixture containing a large number of contaminants.

  Accordingly, an object of the present invention is to provide a method for patterning a plurality of types of cells on a substrate quickly and easily. Furthermore, the present invention provides a method that can be applied not only to patterning cells but also to other lipid membrane-containing materials (for example, organelles, vesicles, viruses, liposomes, micelles, and lipid-coated quantum dots). Is an issue. Another object of the present invention is to provide a method for isolating a specific lipid membrane-containing material.

  As a result of intensive investigations in view of the above problems, the present inventors have developed a novel compound having a function of immobilizing lipid membrane-containing materials including cells by light irradiation. It was revealed that the base material modified with the compound on the surface can rapidly immobilize arbitrary cells without the need for spontaneous adhesion of cells by light irradiation. On the other hand, cells are not immobilized on the surface of the substrate that has not been irradiated with light. The present inventors have also found that a plurality of types of cells can be patterned quickly and easily by repeating light irradiation and cell seeding on the surface of the substrate. Furthermore, the present inventors have found that a specific lipid membrane-containing material can be selectively isolated by using the substrate. The present invention is based on the above findings.

That is, this invention has the following aspects.
[1]
The following formula (I)
[Where:
A ¹ is a lipid membrane-binding group that binds to the lipid membrane in the lipid membrane-containing material,
A ² is a lipid membrane binding inhibitor that inhibits the binding of the lipid membrane binding group to the lipid membrane;
A ³ is a photoreactive group,
A ⁴ is a hydrophilic group,
A ⁵ is a reactive group to the substrate to be modified,
A ⁶ is a scaffold site having at least three bonds,
L ¹ and L ² are linkers;
k ¹ and k ² are each independently 0 or 1]
The compound represented by the above, wherein the inhibition of binding by the lipid membrane binding inhibitory group is eliminated by a photoreaction, and the lipid membrane binding group can bind to the lipid membrane.
[2]
The compound according to [1] above, wherein the lipid membrane-containing material is selected from the group consisting of cells, organelles, vesicles, viruses, liposomes, micelles, and quantum dots coated with lipids.
[3]
[1] or [1] above, wherein the substrate to be modified is a substrate coated with at least one selected from the group consisting of bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTES) and collagen The compound according to [2].

[4]
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Any one of the above [1] to [3], selected from the group, wherein adjacent carbon atoms in the C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds Compounds described in 1.
[5]
A ³ is selected from the group consisting of 2-nitrobenzyl skeleton, coumarin-4-ylmethyl skeleton, phenylcarbonylmethyl skeleton, 7-nitroindolinocarbonyl skeleton, azobenzene skeleton, fulgide skeleton, spiropyran skeleton, spirooxazine skeleton and diarylethene skeleton The compound as described in any one of the above [1] to [4], which is a divalent group having a skeleton.
[6]
A ⁴ is selected from the group consisting of:
[Where:
Z is an oxyalkylene group having 2 to 4 carbon atoms, n represents the average number of added moles of the oxyalkylene group having 2 to 4 carbon atoms, and 2 ≦ n ≦ 500,
The left arrow indicates the connection to A ⁶ , the right arrow indicates the connection to A ⁵ when k ² is 0, and the connection to L ² when k ² is 1]
The compound as described in any one of the above-mentioned [1] to [5] represented by
[7]
A ⁵ is the following substituent:
[Where:
The arrows indicate the connection to A ⁴ when k ² is 0, the connection to L ² when k ² is 1,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group]
The compound according to any one of [1] to [6], selected from the group consisting of:
[8]
L ¹ and L ² are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _{1 -10} alkyl), O or S, the compound according to any one of the above [1] to [7].
[9]
A ⁶ is selected from the group consisting of:
[Where:
L ³ , L ⁴ and L ⁵ are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms among the carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), _optionally substituted with O or S,
k ³ , k ⁴ and k ⁵ are each independently 0 or 1,
Arrows to (A ¹ ), (A ³ ), and (A ⁴ ) indicate connections to A ¹ , A ^3, and A ⁴ , respectively]
The compound according to any one of [1] to [8], represented by:
[10]
The following formula (Ia):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group wherein adjacent carbon atoms in said C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
L ^{1 to} L ⁵ are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _{1 -10} alkyl), O or S may be substituted,
A ³ is a divalent group having a skeleton selected from the group consisting of a nitrobenzene skeleton, a coumarin skeleton, a hydroxyphenanthyl skeleton, a nitroindoline skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton. ,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to L ²
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound as described in any one of the above-mentioned [1] to [9] represented by:
[11]
The following formula (Ib):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group wherein adjacent carbon atoms in said C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
n represents the average number of moles added of oxyethylene groups, and 45 ≦ n ≦ 500,
p is 0-8,
q and s are each independently 0 to 7,
r is 0-10,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to the rest of the compound,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound as described in any one of the above-mentioned [1] to [10] represented by:
[12]
The following formula (Ic)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound according to any one of [1] to [11], represented by:
[13]
The following formula (II-a)
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group, wherein adjacent carbon atoms in the C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
n represents the average number of moles added of oxyethylene groups, and 45 ≦ n ≦ 500,
p is 0-8,
q is each independently 0 to 7,
r is 0-10,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to the rest of the compound,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound as described in any one of [1]-[10] represented by these.
[14]
The following formula (II-b)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
[13] The compound according to [13].
[15]
The following formula (Id)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
Or the following formula (II-c)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound as described in any one of [1]-[10] represented by these.
[16]
A base material for immobilizing a lipid membrane-containing material obtained by reacting the compound according to any one of [1] to [15] above with a base material to be modified.
[17]
The group of formula (III) below:
[Where:
A ^{1 to} A ⁴ , A ⁶ , L ¹ , L ² , k ¹ and k ² are as defined in any one of [1] to [15],
A ⁷ is a divalent group that links the other part of formula (III) and the substrate to be modified, or a reactive group that can be linked to the substrate by a non-covalent bond,
Arrow indicates covalent or non-covalent linkage to substrate]
A substrate for immobilizing a lipid membrane-containing material.
[18]
A method for patterning a lipid membrane-containing material on a substrate, comprising the following steps:
(1) A portion of the lipid membrane-containing material forming the pattern of the lipid membrane-containing material is irradiated with light on the substrate for immobilizing the lipid membrane-containing material according to [16] or [17], and the lipid membrane of the part Making the binding group capable of binding to the lipid membrane;
(2) The method comprising reacting the lipid membrane-containing material with the lipid membrane-binding group to form a pattern of the lipid membrane-containing material.
[19]
The following steps:
(3) The substrate obtained in step (2) is washed to remove unreacted lipid membrane-containing material from the substrate;
(4) Using a lipid membrane containing material different from the lipid membrane containing material used in the previous step, repeating steps (1) to (3) to form a plurality of different lipid membrane containing patterns The method according to [18], further comprising:
[20]
A method for isolating a lipid membrane-containing material comprising the following steps:
(1) Applying a sample containing a lipid membrane-containing material on a substrate for immobilizing the lipid membrane-containing material according to [16] or [17];
(2) detecting the position of the lipid membrane-containing material on the substrate based on microscopic observation;
(3) irradiating the position with light so that the lipid membrane-bound group at the position can be bound to the lipid membrane, and immobilizing the lipid membrane-containing material on the substrate;
(4) The said method including wash | cleaning the base material obtained at step (3), and removing the substance which is not fix | immobilized in the base material from a base material.

  According to the present invention, not only a single type of cell but also a plurality of types of cells can be patterned quickly and easily. The present invention is also applicable to patterning not only cells but also other lipid membrane-containing materials (for example, organelles, vesicles, viruses, liposomes, micelles and lipid-coated quantum dots). Furthermore, it is possible to selectively isolate a specific lipid membrane-containing material.

FIG. 1 shows a photomask for producing a high-density single cell array. FIG. 2 is a photomicrograph (fluorescence, bright field, x4 objective lens) of an array in which Ba / F3 cells are arranged at high density on a substrate. FIG. 3 shows the procedure of a method for preparing a single cell array of a plurality of cell populations. FIG. 4 is a photomicrograph of a single cell array of multiple cell populations fluorescently labeled with EGFP and Cytored. FIG. 5 shows the flow channel device used in Example 5. FIG. 6 shows multiple cell population line patterning. FIG. 7 shows human patterning of multiple cell populations. FIG. 8 shows the evaluation results of cell proliferation and cell viability in cell culture on the substrate surface modified with compound (Ic). FIG. 9 is a photomicrograph of single cell array patterning of adherent cells (HepG2 cells). FIG. 10 is a photomicrograph of single cell array patterning of primary cultured cells (NHDF cells). FIG. 11 is a photomicrograph of array patterning of liposomes labeled with rhodamine. FIG. 12 shows human patterning of biotin modified lipid coated quantum dots. FIG. 13 shows human patterning of lipid-coated quantum dots modified with carboxylic acid. FIG. 14 shows co-line patterning of non-adherent cells (Ba / F3 (KO)) and adherent cells (HEK293T (EGFP)). FIG. 15 shows co-line patterning of rhodamine labeled liposomes and adherent cells (HEK293T (EGFP)). FIG. 16 shows that circulating cancer cells (CTC) in the blood were captured and isolated in the microchannel coated with compound (Ic). FIG. 17 is a photomicrograph showing that Ba / F3 cells can be immobilized on a substrate modified with compound (II-b) by light irradiation. FIG. 18 shows the evaluation results of substituents that can function as lipid membrane binding inhibitors.

In the present specification, the “C _7-22 alkyl group” is a linear or branched alkyl group having 7 to 22 carbon atoms. Examples of C _7-22 alkyl groups include heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl (stearyl), Nonadecyl group, icosyl group, henicosanyl group, docosanyl group and isomers thereof may be mentioned.

In the present specification, the “C _1-10 alkyl group” is a linear or branched alkyl group having 1 to 10 carbon atoms. Examples of C _1-10 alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and isomers thereof.

In the present specification, the “C _1-10 alkoxy group” is a group in which the above C _1-10 alkyl group is substituted on an oxygen atom. Examples of C _1-10 alkoxy groups include methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy and isomers thereof. Can be mentioned.

  In the present specification, “halogen” is a monovalent group of a halogen atom, and specific examples thereof include a fluoro group, a chloro group, a bromo group, and an iodo group.

In the present specification, in the C _7-22 alkyl group, “adjacent carbon atoms may be linked by 1 to 3 unsaturated bonds” means a straight or branched chain having 7 to 22 carbon atoms. It shows that 1 to 3 unsaturated bonds may be contained in the chain alkyl group. Here, the unsaturated bond is either a double bond or a triple bond. Examples of C _7-22 alkyl groups in which adjacent carbon atoms are linked by 1 to 3 unsaturated bonds include, for example, cis-9-hexadecen-1-yl (palmitoleyl) group, trans-9-octadecenyl (Elidyl) group, cis-9-octadecenyl (oleyl) group, cis, cis-9,12-octadecadienyl (linolenyl) group, (9E, 12E, 15E) -octadeca-9,12,15-trienyl ( Elidelinolenyl) group and the like.

In the present specification, the “C _6-14 aryl group” refers to an aromatic cyclic group having 6 to 14 carbon atoms, and specific examples include a phenyl group, a naphthyl group, and an anthracenyl group.

In the present specification, the "C _6-14 aryl C _7-22 alkyl group", a substituted moiety of the "C _6-14 aryl group" is substituted with the above "C _6-14 aryl group" group It is. Examples of C _6-14 aryl C _7-22 alkyl groups include phenylheptyl, phenyloctyl, phenylnonyl, phenyldecyl, phenylundecyl, phenyldodecyl, phenyltridecyl, phenyltetradecyl , Phenylpentadecyl group, phenylhexadecyl group, phenylheptadecyl group, phenyloctadecyl group, phenylnonadecyl group, phenylicosyl group, phenylhexicosanyl group, phenyldocosanyl group, naphthylheptyl group, naphthyloctyl group, naphthyl Nonyl, naphthyldecyl, naphthylundecyl, naphthyldodecyl, naphthyltridecyl, naphthyltetradecyl, naphthylpentadecyl, naphthylhexadecyl, naphthylheptadecyl, naphthyloctadecyl, naphthylnonadecyl Group, naphthyl icosyl group, naphthyl henicosanyl group, naphthyl docosanyl group, anthracenyl heptyl group, anthracenyl octyl group, anthracenyl nonyl group, anthracenyl decyl group, anthracenyl undecyl group, anthracenyl Dodecyl group, anthracenyl tridecyl group, anthracenyl tetradecyl group, anthracenyl pentadecyl group, anthracenyl hexadecyl group, anthracenyl heptadecyl group, anthracenyl octadecyl group, anthracenyl nonadecyl group, Anthracenyl icosyl group, anthracenyl henicosanyl group, anthracenyl docosanyl group, etc. are mentioned.

In the present specification, the “C _7-22 alkyl C _6-14 aryl group” is a group in which a substitutable portion of the “C _6-14 aryl group” is substituted with the “C _7-22 alkyl group”. It is. Examples of C _7-22 alkyl C _6-14 aryl groups include heptylphenyl, octylphenyl, nonylphenyl, decylphenyl, undecylphenyl, dodecylphenyl, tridecylphenyl, tetradecylphenyl , Pentadecylphenyl group, hexadecylphenyl group, heptadecylphenyl group, octadecylphenyl group, nonadecylphenyl group, icosylphenyl group, henicosanylphenyl group, docosanylphenyl group, heptylnaphthyl group, octylnaphthyl group, nonyl Naphthyl, decylnaphthyl, undecylnaphthyl, dodecylnaphthyl, tridecylnaphthyl, tetradecylnaphthyl, pentadecylnaphthyl, hexadecylnaphthyl, heptadecylnaphthyl, octadecylnaphthyl, nonadecylnaphthyl Group, icosylnaphthyl group, henicosanylnaphthyl group, docosanylnaphthyl group, heptylanthracenyl group, octylanthracenyl group, nonylanthracenyl group, decylanthracenyl group, undecylanthracenyl group, dodecylanthracyl group Cenyl group, tridecylanthracenyl group, tetradecylanthracenyl group, pentadecylanthracenyl group, hexadecylanthracenyl group, heptadecylanthracenyl group, octadecylanthracenyl group, nonadecylanthracenyl group Group, icosylanthracenyl group, henicosanylanthracenyl group, docosanylanthracenyl group and the like.

  As used herein, “oxo group” refers to a group that forms a carbonyl group together with the carbon atom to which it is attached.

  In the present specification, examples of the “oxyalkylene group having 2 to 4 carbon atoms” include an oxyethylene group, an oxypropylene group, an oxytrimethylene group, an oxybutylene group, and an oxytetramethylene group.

In the present specification, the “C _6-14 arylene group” means an arylene group having 6 to 14 carbon atoms. Specific examples include a phenylene group, a naphthylene group, and an anthranylene group.

In the present specification, the “C _1-10 alkylene group” means a linear or branched alkylene group having 1 to 10 carbon atoms. Specific examples include methylene group, ethylene group, propylene group, butylene group, pentylene group, hexylene group, heptylene group, octylene group, nonylene group, decalene group and isomers thereof.

In the present specification, for the C _1-10 alkylene group, “the carbon atom in the alkylene group may be substituted with 1 to 5 oxo groups, and adjacent carbon atoms are 1 to 5 unsaturated bonds. And 1 to 4 of the carbon atoms in the alkylene group may be replaced by NH, N (C _1-10 alkyl), O or S ". Indicates that, for example, the following structure can be taken.
[Where:
m1 is 0-6, m2 is 0-7, m3 is 0-8, m4 is 0-9, m5 is 0-10, NH is optionally N (C _{1- 10} alkyl). ]

The present invention provides the following formula (I)
[Where:
A ¹ is a lipid membrane-binding group that binds to the lipid membrane in the lipid membrane-containing material,
A ² is a lipid membrane binding inhibitor that inhibits the binding of the lipid membrane binding group to the lipid membrane;
A ³ is a photoreactive group,
A ⁴ is a hydrophilic group,
A ⁵ is a reactive group to the substrate to be modified,
A ⁶ is a scaffold site having at least three bonds,
L ¹ and L ² are linkers;
k ¹ and k ² are each independently 0 or 1]
Wherein the inhibition of binding by the lipid membrane binding inhibitory group is eliminated by photoreaction, and the lipid membrane binding group can bind to the lipid membrane (hereinafter referred to as “the compound of the present invention”). ").

  In the present specification, the “lipid membrane-containing material” refers to a substance containing an arbitrary lipid membrane. Here, “lipid membrane” refers to a membrane-like lipid. In the present specification, “lipid” refers to a group of substances that are hardly soluble in water and easily soluble in organic solvents. Lipids typically include long chain fatty acids and derivatives or analogs thereof, but herein also includes a group of organic compounds such as steroids, carotenoids, terpenoids, isoprenoids, and fat-soluble vitamins. Examples of lipids include: 1) simple lipids (esters of fatty acids and alcohols, also called neutral lipids), for example, fats and oils (triacylglycerol), waxes (waxes, fatty acid esters of higher alcohols), sterol esters, fatty acids of vitamins 2) Complex lipids (compounds having ester bonds or amide bonds and having polar groups in addition to fatty acids and alcohols such as phosphoric acid, sugar, sulfuric acid, amines, etc.) Glycerophospholipids and sphingophospholipids), glycolipids (including glyceroglycolipids and glycosphingolipids), lipoproteins, sulfolipids, etc .; 3) derived lipids (compounds produced by hydrolysis of simple and complex lipids) Of these, fat-soluble ones, fatty acids, higher alcohols, fat-soluble vitamins, Steroids, such as a hydrocarbon is included) include, but are not limited to.

  Although it does not specifically limit as a lipid membrane containing material, For example, the structure (For example, the quantum dot coat | covered with a cell, an organelle, a vesicle, a virus, a liposome, a micelle, and a lipid) is mentioned. Examples of cells include animal cells, plant cells converted to protoplasts, and microorganisms. The cell may be an adherent cell or a non-adherent cell.

In the present specification, the “lipid membrane binding group” is a group that binds non-covalently to the lipid membrane of the lipid membrane-containing material. The lipid membrane binding group is not particularly limited. For example, a C _7-30 alkyl group (preferably a C _7-22 alkyl group), a C _6-14 aryl group, a C _6-14 aryl C _7-30 alkyl group (preferably _Are hydrophobic groups such as C _6-14 aryl C _7-22 alkyl group) and C _7-30 alkyl C _6-14 aryl group (preferably C _6-14 aryl C _7-22 alkyl group). Here, adjacent carbon atoms in the C _7-30 alkyl group may be linked by 1 to 3 unsaturated bonds. As the lipid membrane binding group, a hydrophobic group is preferable. As the lipid membrane binding group, adjacent good C _7-30 alkyl group carbon atoms are linked by one to three unsaturated bonds, linked by neighboring 1-3 unsaturated bonds carbon atoms An _{optionally substituted} C _7-22 alkyl group, or a C _11-22 alkyl group in which adjacent carbon atoms may be linked by 1 to 3 unsaturated bonds, or 1 to 3 adjacent carbon atoms A C _16-18 alkyl group which may be linked by an unsaturated bond is more preferable. Examples of lipid membrane binding groups include hexadecyl group, heptadecyl group, octadecyl (stearyl) group, cis-9-hexadecenyl (palmitoleyl) group, cis-8-heptadecenyl group, trans-8-heptadecenyl group, trans-9-octadecenyl group. (Elidyl) group, cis-9-octadecenyl (oleyl) group, cis, cis-9,12-octadecadienyl (linolenyl) group, (9E, 12E, 15E) -octadeca-9,12,15-trienyl ( Elidelinolenyl) group is more preferable, and cis-8-heptadecenyl group and oleyl group are particularly preferable.

In the present specification, the “lipid membrane binding inhibitory group” is a group that physically or chemically inhibits the binding of a lipid membrane binding group to a lipid membrane. The structure of the lipid membrane binding inhibitor group may be the same as or different from the structure of the lipid membrane binding group. The lipid membrane binding inhibitor group is not particularly limited, but for example, a C _7-30 alkyl group (preferably a C _7-22 alkyl group), a C _6-14 aryl group, a C _6-14 aryl C _7-30 alkyl group ( C _6-14 aryl C _7-22 alkyl group), and C _7-30 alkyl C _6-14 aryl group (preferably C _7-22 alkyl C _6-14 aryl group), hydrophobic groups such as cholesteryl group Is mentioned. Here, adjacent carbon atoms in the C _7-30 alkyl group may be linked by 1 to 3 unsaturated bonds. As the lipid membrane binding inhibitory group, a hydrophobic group is preferable. The lipid membrane binding inhibitory group includes a C _7-30 alkyl group in which adjacent carbon atoms may be linked by 1 to 3 unsaturated bonds, and an adjacent carbon atom by 1 to 3 unsaturated bonds. A C _7-22 alkyl group that may be linked, or a C _11-22 alkyl group that may have adjacent carbon atoms connected by 1 to 3 unsaturated bonds, or 1 to 3 carbon atoms More preferred are C _16-18 alkyl groups which may be linked by one unsaturated bond. Examples of the lipid membrane binding inhibiting group include hexadecyl group, heptadecyl group, octadecyl (stearyl) group, cis-9-hexadecenyl (palmitoleyl) group, cis-8-heptadecenyl group, trans-8-heptadecenyl group, trans-9- Octadecenyl (elaidyl) group, cis-9-octadecenyl (oleyl) group, cis, cis-9,12-octadecadienyl (linolenyl) group, (9E, 12E, 15E) -octadeca-9,12,15-trienyl (Elidelinolenyl) group is more preferable, and cis-8-heptadecenyl group and oleyl group are particularly preferable.

  In the present specification, the “photoreactive group” refers to a group in which the bond in the photoreactive group is cleaved by the photoreaction or the structure thereof is changed.

  The physical or chemical binding inhibition by the lipid membrane binding inhibitory group is eliminated by the photoreaction, and the lipid membrane binding group in the compound of the present invention can bind to the lipid membrane. In one embodiment, the photoreactive group is excised from the scaffold site by photoreaction while remaining linked to the lipid membrane binding inhibitory group. Thereby, the binding inhibition by the lipid membrane binding inhibitory group is eliminated. The photoreactive group that can be used in the present embodiment is not particularly limited as long as it is cut out from the scaffold site. For example, a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, or a 7-nitroindolinocarbonyl skeleton Is a divalent group.

In the present specification, the “divalent group having a 2-nitrobenzyl skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the arrow on the right side indicates the connection to the scaffold site, the arrow on the left side indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. L ⁶ is an ethynylene group or absent. ]

The following are preferable as the divalent group having a 2-nitrobenzyl skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a coumarin-4-ylmethyl skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. L ⁷ is a C _1-10 alkylene group or is not present, wherein the carbon atom in the alkylene group may be substituted with 1 to 5 oxo groups, and adjacent carbon atoms 1 to 5 may be connected to each other by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms among the carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), O or S may be substituted. ]

The following are preferable as the divalent group having a coumarin-4-ylmethyl skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a phenylcarbonylmethyl skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

The following are preferable as the divalent group having a phenylcarbonylmethyl skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a 7-nitroindolinocarbonyl skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

As the divalent group having a 7-nitroindolinocarbonyl skeleton, the following are preferable.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

  In another embodiment, the structure of the photoreactive group is changed by the photoreaction, and the distance between the lipid membrane binding inhibitor and the lipid membrane binding group linked to the photoreactive group is increased accordingly. Thereby, the binding inhibition by the lipid membrane binding inhibitory group is eliminated. The photoreactive group that can be used in the present embodiment is not particularly limited as long as the photoreaction eliminates the binding inhibition by the lipid membrane binding inhibitory group. For example, an azobenzene skeleton, fulgide skeleton, spiropyran skeleton, spirooxazine skeleton, or It is a divalent group having a diarylethene skeleton.

In the present specification, the “divalent group having an azobenzene skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. L ⁸ is a C _1-10 alkylene group or is not present, wherein the carbon atom in the alkylene group may be substituted with 1 to 5 oxo groups, Atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms among the carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), O Or it may be replaced by S. ]

The following are preferable as the divalent group having an azobenzene skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a fulgide skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

The following are preferable as the divalent group having a fulgide skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a spiropyran skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

As the divalent group having a spiropyran skeleton, the following skeletons are preferable.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a spirooxazine skeleton” is a divalent group having the following structure or a derivative structure thereof.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

The following are preferable as the divalent group having a spirooxazine skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

In the present specification, the “divalent group having a diarylethene skeleton” is a divalent group having the following structure or a derivative structure thereof.
[Wherein X is S, NH, N (C _1-10 alkyl) or O, the arrow on the right side indicates connection to the scaffold site, and the arrow on the left side indicates that A ² when k ¹ is 0. When k ¹ is 1, it indicates a connection to L ¹ , and k ⁶ is 1-6. ]

The following are preferable as the divalent group having a diarylethene skeleton.
[In the formula, the right arrow indicates the connection to the scaffold site, the left arrow indicates the connection to A ² when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates. ]

  In the present specification, the “hydrophilic group” is a divalent group containing a water-soluble compound residue, preferably a divalent group containing a water-soluble polymer compound residue. Although the compound which gives such a residue is not specifically limited, For example, polyalkylene glycol, polysaccharide, polylactic acid, polyvinyl alcohol, polypeptide, polyacrylic acid, polyacrylamide etc. are mentioned.

The “hydrophilic group” of the compounds of the present invention preferably has the following structure:
[Where:
Z is an oxyalkylene group having 2 to 4 carbon atoms, n represents the average number of added moles of the oxyalkylene group having 2 to 4 carbon atoms, and 2 ≦ n ≦ 500,
The left arrow indicates a connection to A ⁶ , the right arrow indicates a connection to A ⁵ when k ² is 0, and a connection to L ² when k ² is 1. ]

In the above formula, Z is preferably an oxyethylene group. That is, the hydrophilic group is preferably one of the following two structures.
[Where:
Z represents the average number of added moles of the oxyalkylene group having 2 to 4 carbon atoms, and 2 ≦ n ≦ 500,
The left arrow indicates a connection to A ⁶ , the right arrow indicates a connection to A ⁵ when k ² is 0, and a connection to L ² when k ² is 1. ]

  In the above formula, there are as many oxyalkylene groups as n, but these oxyalkylene groups may be used alone or in combination of two or more. When two or more kinds are combined, there is no restriction on the combination. The oxyalkylene group may be in a block form or a random form. When the ratio of oxyethylene groups to all oxyalkylene groups is 50 to 100 mol%, it is preferable in that good water solubility is obtained.

  In the above formula, n is preferably 45 ≦ n ≦ 500, and more preferably 45 ≦ n ≦ 200.

In the present specification, the “reactive group to the substrate to be modified” (hereinafter, also simply referred to as “reactive group”) means that the present invention reacts covalently or non-covalently with the substrate to be modified. This is a group for immobilizing the compound on the substrate to be modified. When the reaction between the reactive group and the base material to be modified is a covalent bond, the base material to be modified has at least one functional group that can be covalently bonded to the reactive group. Examples of such covalently bondable functional groups include the following.
[In the formula, the arrow indicates the connection with the base material to be modified. ]

The covalent bond “reactive group to the substrate to be modified” is not particularly limited as long as it can react with a functional group contained in the substrate to be modified to form a covalent bond, and examples thereof include the following substituents. :
[Where:
The arrows indicate the connection to A ⁴ when k ² is 0, the connection to L ² when k ² is 1,
X is halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of C _1-10 alkyl, halogen, C _1-10 alkoxy. ]
Among these, a carboxyl group and the following substituents are preferable.

As an example of immobilizing the “reactive group to the base material to be modified” (hereinafter also simply referred to as “reactive group”) of the compound of the present invention by reacting non-covalently with the base material to be modified, for example, Is mentioned.
(I) When a hydrophobic substituent is employed as a reactive group of the compound of the present invention and is immobilized by a hydrophobic interaction with a substrate to be modified containing the hydrophobic substituent.
(Ii) When a polypeptide or nucleic acid is employed as a reactive group of the compound of the present invention and is immobilized by reacting with a base material to be modified containing a substance capable of binding thereto (for example, a combination of complementary DNA strands; Combination of biotin and avidin).
(Iii) A case where a small molecule (for example, ferrocene) capable of recognizing a supramolecule (for example, cyclodextrin) is employed as a reactive group of the compound of the present invention, and is reacted with a substrate to be modified containing the supramolecule and immobilized.

  In the present specification, the “substrate to be modified” of the compound of the present invention is not particularly limited. For example, the substrate (where the compound of the present invention is to be modified is a plate or film, for example, a slide glass) , Dishes, microplates, microarray substrates), carriers (eg, beads), fibrous structures, tubes, containers (eg, test tubes and vials), and the like. Materials for the base material to be modified include glass; cement; ceramics such as ceramics or fine ceramics; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate; biomaterials such as polypeptides and proteins; silicon; Examples include activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; non-woven fabric; filter paper; membrane filter; Since the surface of the base material to be modified introduces amino groups, carboxyl groups, hydroxy groups, etc., it can be coated with a polymer such as polycation or treated with a silane coupling agent having a substituent introduced onto the surface of the base material. It may be given. Alternatively, a reactive functional group may be introduced by plasma treatment. As the “substrate to be modified” of the compound of the present invention, a substrate coated with at least one selected from the group consisting of bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTES) and collagen is preferable. .

In the compound of the present invention, a linker L ¹ may be present between A ² and A ^3, and a linker L ² may be present between A ⁴ and A ⁵ . In the present specification, the “linker” is a divalent group for connecting two functional groups. Although it does not specifically limit as a linker, For example, they are a _C6-14 arylene group or a _C1-10 alkylene group. Here, carbon atoms in the alkylene group may be substituted with 1 to 5 oxo groups, adjacent carbon atoms may be connected with 1 to 5 unsaturated bonds, and the alkylene Among the carbon atoms in the group, 1 to 4 carbon atoms may be replaced with NH, N (C _1-10 alkyl), O or S.

In the compounds of the present invention, between L ¹ and A ² and between L ¹ and A ³ , and between L ² and A ⁴ and between L ² and A ⁵ are each independently an alkylene structure, amide It is preferable to have a structure selected from the group consisting of a structure, an ester structure, an amino structure, and an ether structure.

Examples of the “linker” L ¹ and L ² include the following formula:
[Where:
m1 is 0-6, m2 is 0-7, m3 is 0-8, m4 is 0-9, m5 is 0-10, NH is optionally N (C _{1- 10} alkyl) may be substituted]
It preferably has a structure represented by

In this specification, the “scaffold site” refers to a chemical structure having at least three bonds. The said scaffold site | part couple | bonds with a lipid membrane binding group, a photoreactive group, and a hydrophilic group, The compound of this invention is formed. Although it does not specifically limit as a scaffold site | part, For example, it has the following structures:
[Where:
L ³ , L ⁴ and L ⁵ are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms among the carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), _optionally substituted with O or S,
k ³ , k ⁴ and k ⁵ are each independently 0 or 1,
The arrows to (A ¹ ), (A ³ ) and (A ⁴ ) indicate the connection to A ¹ , A ³ and A ⁴ , respectively. ]

In the above formula, between L ³ and A ¹ and between L ⁴ and A ⁴ are each independently selected from the group consisting of an alkylene structure, an amide structure, an ester structure, an amino structure, and an ether structure. It preferably has a structure.

In the above formula, as L ³ , L ⁴ and L ⁵ , for example, the following formula:
[Where:
m1 is 0-6, m2 is 0-7, m3 is 0-8, m4 is 0-9, m5 is 0-10, NH is optionally N (C _{1- 10} alkyl) may be substituted]
It preferably has a structure represented by

As a scaffold site, preferably
[Where:
L ³ is
(Where
m3 is 0-8,
(The arrow on the left indicates the connection to the lipid membrane binding group, and the arrow on the right indicates the connection to the central carbon of the scaffold site)
And
L ⁴ is
(Where
m2 is 0-7,
(The arrow on the left indicates the connection to the central carbon of the scaffold site, and the arrow on the right indicates the connection to the hydrophilic group)
And
L ⁵ is NH]
It is.

As preferred compounds of formula (I), the following formula (Ia):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group wherein adjacent carbon atoms in said C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
L ^{1 to} L ⁵ are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _{1 -10} alkyl), O or S may be substituted,
A ³ is a divalent group having a skeleton selected from the group consisting of a nitrobenzene skeleton, a coumarin skeleton, a hydroxyphenanthyl skeleton, a nitroindoline skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton. ,
A ⁵ is the following substituent:
[Where:
The arrow indicates the connection to L ²
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of C _1-10 alkyl, halogen, C _1-10 alkoxy)
Selected from the group consisting of]
The compound represented by these is mentioned.

As more preferred compounds of formula (I), the following formula (Ib):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group, and a C _7-22 alkyl C _6-14 aryl group. Selected from the group wherein adjacent carbon atoms in said C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
n represents the average number of moles added of oxyethylene groups, and 45 ≦ n ≦ 500,
p is 0-8,
q and s are each independently 0 to 7,
r is 0-10,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to the rest of the compound,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of C _1-10 alkyl, halogen, C _1-10 alkoxy)
Selected from the group consisting of]
The compound represented by these is mentioned.

As further preferred compounds of formula (I), the following formula (Ic):
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound represented by these is mentioned.

Moreover, as a preferable compound of the formula (I), the following formula (II-a):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group, wherein adjacent carbon atoms in the C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
n represents the average number of moles added of oxyethylene groups, and 45 ≦ n ≦ 500,
p is 0-8,
q is each independently 0 to 7,
r is 0-10,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to the rest of the compound,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound represented by these is mentioned.

As preferred compounds of formula (II-a), the following formula (II-b):
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound represented by these is mentioned.

In addition, preferred compounds of formula (I) include the following compounds of formula (Id) and compounds of formula (II-c).
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500. ]
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500. ]

General Production Method The compound represented by the formula (I) can be synthesized by adapting various known synthesis methods using characteristics based on the basic skeleton or the type of substituent. Although the typical manufacturing method is illustrated below, it is not limited only to the method as described below. Depending on the type of functional group, it may be effective in terms of production technology to change the functional group to a suitable protecting group at the raw material or intermediate stage, that is, a group that can be easily converted to the functional group. Yes, the protecting group can be removed as necessary to obtain the desired compound. Examples of such a functional group include a hydroxyl group, a carboxyl group, an amino group, and the like, and examples of the protecting group include Protective Group 3 Organic Synthesis 3rd edition “Protective” by Green and Wutt. Protecting groups described in “Groups in Organic Synthesis (third edition)” can be used, and these may be appropriately used depending on the reaction conditions. The starting materials and the reaction reagents are known compounds, or can be easily produced from known compounds according to methods well known in the field of organic chemistry.

The compounds of formula (Ib) included in formula (I) can be prepared as outlined in Scheme 1 below. The symbols in the following scheme are the same as those described above.

Process 1
Compound 3 can be prepared by reacting the amino group of compound 1 with compound 2, which is an N-hydroxysuccinimide ester. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. N, N-dimethylformamide, tetrahydrofuran and acetonitrile are preferred. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 7 days, preferably 1 hour to 72 hours.

This step can also be performed using an activated ester obtained by using another activator instead of the N 1 -hydroxysuccinimide ester of A ¹ -C (O) OH. Such activators are not particularly limited and include, for example, N-hydroxysulfosuccinimide, 4-nitrophenyl chloroformate, 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole.

Process 2
The compound 4 which is N-hydroxysuccinimide ester can be manufactured by making the carboxyl group of the compound 3 react with N-hydroxysuccinimide. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Preferred is dichloromethane. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 3 days, preferably 1 hour to 24 hours.

  In Step 2, the carboxyl group of Compound 3 can be subjected to the subsequent Step 3 using an activated ester obtained by using another activating agent instead of the N-hydroxysuccinimide ester. It can also be done. Such activated esters are not particularly limited, and examples thereof include N-hydroxysulfosuccinimide, 4-nitrophenyl chloroformate, 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole.

Process 3
Compound 6 can be produced by reacting compound 4 which is an N-hydroxysuccinimide ester with the amino group of compound 5. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Preferred is dichloromethane. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 3 days, preferably 1 hour to 24 hours.

Process 4
The Boc group, which is a protecting group for the amino group of Compound 6, can be deprotected under acidic conditions to give Compound 7. The acid used in this step is not particularly limited, and examples thereof include hydrochloric acid, sulfuric acid, nitric acid, formic acid, trifluoroacetic acid, methanesulfonic acid, and p-toluenesulfonic acid. Preferred is trifluoroacetic acid or hydrochloric acid. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 2-propanol, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane , Cyclohexane, N, N-dimethylformamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Preferred is dichloromethane or methanol. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 3 days, preferably 1 hour to 24 hours.

Process 5
Compounds 9 and 8 can be condensed in the presence of a base to produce compound 9. The base used in this step is not particularly limited, and examples thereof include trimethylamine, triethylamine, triisopropylamine, diisopropylethylamine, tributylamine, pyridine, and 4-dimethylaminopyridine. Trimethylamine or triethylamine is preferable. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Preferred is dichloromethane. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 7 days, preferably 12 hours to 72 hours.

Step 6
The compound of formula (Ib) having a reactive group A5 on the base material to be modified can be produced by activating the carboxyl group of compound 9 with an activating agent. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Preferred is dichloromethane. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 3 days, preferably 1 hour to 24 hours. For example, when A5 is N-hydroxysuccinimide ester, it can be produced in the same manner as in Step 2 above.

Compound 8 in Scheme 1 above can be prepared, for example, according to Scheme 2 below.

Step 7
Compound 12 can be produced by reacting the carboxyl group of compound 10 with the amino group of compound 11 in the presence of a condensing agent. The condensing agent used in this step is not particularly limited. For example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, benzotriazol-1-yloxy-trisdimethylamino Phosphonium salt, hexafluorophosphoric acid (benzotriazol-1-yloxy) tripyrrolidinophosphonium, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride, etc. Is mentioned. Preferred is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Tetrahydrofuran or dichloromethane is preferred. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 hour to 3 days, preferably 1 hour to 24 hours.

Process 8
The compound of formula 8 can be prepared by reacting the hydroxyl group of compound 12 with 4-nitrophenol chloroformate in the presence of a base. The base used in this step is not particularly limited, and examples thereof include trimethylamine, triethylamine, triisopropylamine, diisopropylethylamine, tributylamine, pyridine, and 4-dimethylaminopyridine. Triethylamine is preferable. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Tetrahydrofuran is preferred. The reaction temperature in this step varies depending on the raw materials and the solvent to be used, but is usually -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 5 minutes to 3 days, preferably 15 minutes to 24 hours.

A compound of formula (II-b) can also be produced by replacing compound 8 in scheme 1 with compound 16 which can be produced according to scheme 3 below.

    Step 9 of Scheme 3 can be performed in the same manner as Step 1 of Scheme 1. Moreover, the said process 10 can be performed similarly to the process 8 of the said scheme 2. Compound 13 can be produced, for example, by the method described in the Examples of the present application.

  In each reaction in the present specification, the reaction involving heating can be performed using a water bath, an oil bath, a sand bath, or microwave, as will be apparent to those skilled in the art.

  In each reaction in the present specification, a solid-phase-supported reagent supported on a high-molecular polymer (for example, polystyrene, polyacrylamide, polypropylene, polyethylene glycol, etc.) may be used as appropriate.

  The compound of formula (I) and each intermediate obtained as described above are isolated and purified by ordinary organic synthesis operations such as extraction, crystallization, recrystallization, dialysis, and various chromatography.

  One embodiment of the present invention is a substrate for immobilizing a lipid membrane-containing material obtained by reacting a compound of the present invention with a substrate to be modified (hereinafter, “substrate of the present invention”). Also called). Although the form of the base material of the present invention is not particularly limited, for example, a substrate (where the compound of the present invention is to be modified is a plate or film, such as a glass slide, a dish, a microplate, a microarray substrate), Examples include carriers (for example, beads), fibrous structures, tubes, containers (for example, test tubes and vials), and the like. Materials for the base material to be modified include glass; cement; ceramics such as ceramics or fine ceramics; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate; biomaterials such as polypeptides and proteins; silicon; Examples include activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; non-woven fabric; filter paper; membrane filter; Since the surface of the base material to be modified introduces amino groups, carboxyl groups, hydroxy groups, etc., it can be coated with a polymer such as polycation or treated with a silane coupling agent having a substituent introduced onto the surface of the base material. It may be given. Alternatively, a reactive functional group may be introduced by plasma treatment.

  In one embodiment, the reaction between the compound of the present invention and the substrate to be modified is performed by bringing the surface of the substrate to be modified into contact with the compound of the present invention. By this contact, the “reactive group to the substrate to be modified” of the compound of the present invention is immobilized by reacting non-covalently with the substrate to be modified, and the substrate for immobilizing the lipid membrane-containing material is Prepared. In the reaction, the type of the solvent used in the reaction between the compound of the present invention and the base material to be modified is not particularly limited as long as it is a solvent that does not participate in the reaction. For example, phosphate buffer, borate buffer, Tris buffer Buffer, acetate buffer, carbonate buffer, buffer such as Good's buffer or isotonic solution thereof; or organic solvent such as acetonitrile, dimethyl sulfoxide, dimethylformamide; or the buffer or isotonic solution and the organic solvent Can be used. In order not to damage or modify the base material to be modified, the compound of the present invention is dissolved in an easily soluble organic solvent (for example, acetonitrile, dimethyl sulfoxide, dimethylformamide) and then sufficiently diluted with the buffer or isotonic solution. Is preferably used. Although reaction temperature is not specifically limited, For example, -78-200 degreeC, Furthermore, 0-100 degreeC is preferable. The reaction time is usually about 1 minute to 72 hours, preferably 30 minutes to 24 hours. After the reaction, the substrate is preferably washed with water.

  In another embodiment, the reaction between the compound of the present invention and the substrate to be modified is performed by mixing the substrate to be modified and the compound of the present invention. The mixing includes, for example, a case where a base material is mixed with the compound of the present invention and a base material is produced using the mixture. In this case, both may react during the mixing step of the base material and the compound of the present invention, or both may react during the manufacturing step of the substrate after the mixing step. The mixing step may be performed by mixing the base material and the compound of the present invention in a solid state, or after dissolving the compound of the present invention in a solvent (for example, the above aqueous solvent and / or organic solvent), You may carry out by mixing the obtained solution and base material. The manufacturing process of a base material can be performed by a general method well known to the manufacturer of the base material.

One aspect of the present invention is a group of the following formula (III):
[Where:
A ^{1 to} A ⁴ , A ⁶ , L ¹ , L ² , k ¹ and k ² are as defined in the compounds of the invention above;
A ⁷ is a divalent group that connects the other part of formula (III) and the substrate, or a reactive group that can be connected to the substrate by a non-covalent bond,
Arrow indicates covalent or non-covalent linkage to substrate]
The present invention relates to a base material for immobilizing a lipid membrane-containing material. Hereinafter, it is also referred to as “a substrate having a group of the formula (III) of the present invention”.

  The form and raw material of the substrate having the group of the formula (III) of the present invention may be those exemplified above as the form and raw material of the “substrate of the present invention”.

  In the base material having the group of the formula (III) of the present invention, the divalent group for linking the other part of the formula (III) and the base material is not particularly limited, but for example, sharing in the compound of the present invention Examples thereof include a divalent group formed by a reaction between a binding “reactive group to the substrate to be modified” and a functional group contained in the substrate to be modified. The covalent bond “reactive group to the substrate to be modified” may be those exemplified above with respect to the compound of the present invention.

  In the substrate having the group of the formula (III) of the present invention, the reactive group that can be linked to the substrate by a non-covalent bond is not particularly limited. For example, the above-mentioned “substrate to be modified” in the compound of the present invention The “reactive group” may be the same as mentioned in the example of immobilizing by reacting non-covalently with the base material to be modified.

  The base material having a group of the formula (III) of the present invention can be obtained, for example, by reacting the compound of the present invention with a base material to be modified. As the reaction conditions, for example, the above-described method for preparing the “substrate of the present invention” can be applied.

  One aspect of the present invention is a method for patterning a lipid membrane-containing material on a substrate, which comprises the following steps: (1) light is applied to a portion of the substrate of the present invention that forms the pattern of the lipid membrane-containing material. Irradiating to make the lipid membrane-bound group of the part capable of binding to the lipid membrane; (2) reacting the lipid membrane-containing material with the lipid membrane-bound group to form a pattern of the lipid membrane-containing material (Hereinafter also referred to as “patterning method of the present invention”).

  In the present specification, “patterning” refers to locally modifying a base material, placing the lipid membrane-containing material at a desired position on the base material, and immobilizing it. In patterning, a single type of lipid membrane-containing material may be arranged in a specific pattern on the substrate, or a plurality of types of lipid membrane-containing materials may be arranged in a specific pattern. Although the shape of a pattern is not specifically limited, For example, you may arrange | position a lipid membrane containing thing with a fixed space | interval. In this case, the number of places where the lipid membrane-containing material is disposed is not limited, and even one point is included in “patterning”. Moreover, the shape of a pattern may arrange | position a lipid membrane containing material continuously.

In the step (1), the wavelength of the light to be irradiated may be determined according to the type of the photoreactive group in the compound of the present invention, and is usually in the range of 157 to 600 nm, preferably in the vicinity of 250 to 450 nm. Irradiate the light. However, in the case of performing a photoreaction by multiphoton absorption, it is possible to use light having a longer wavelength than the above. Light sources such as sunlight, electric light such as mercury lamps, laser light (semiconductor lasers, solid state lasers, gas lasers), light emission of light emitting diodes, light emission of electroluminescent elements can be used. As the light irradiation method, light from the light source can be uniformly irradiated onto the substrate surface through an appropriate filter as necessary, or pattern exposure of a desired shape may be performed using a so-called mask. . Alternatively, the light may be collected using a lens or a mirror and irradiated in a fine shape. Alternatively, the condensed light beam may be subjected to scanning exposure. In the case of pattern exposure, the exposure may be performed by contact exposure, which is an exposure format in which a mask (reticle) and a workpiece (sample) are brought into contact with each other for exposure. Further, proximity exposure, which is a non-contact exposure method in which exposure is performed by setting a gap between a mask (reticle) and a workpiece (sample) to about several μm to several tens of μm, may be performed. Furthermore, you may use the projection exposure method (maskless exposure method) which projects the image produced with the liquid crystal or the digital mirror device on the workpiece | work surface. Although reaction temperature is not specifically limited, Usually, it is -78-200 degreeC, 0-100 degreeC is preferable, and when the aqueous medium containing biological samples, such as a cell, exists on a workpiece | work, 4-50 degreeC is more preferable. . Light irradiation energy should just be a grade which can exhibit the function which the base-material surface modified with this compound fix | immobilizes a lipid membrane containing material, and is 0.001-1000J / cm < ² > normally, 0.01- 100 J / cm ² is preferred.

  The reaction in the above step (2) is usually performed at −78 to 200 ° C., preferably 0 to 100 ° C. When an aqueous medium containing a biological sample such as a cell is present on the workpiece, 4 to 50 ° C. More preferred. Usually, it is performed for 1 second to 120 minutes, preferably 15 seconds to 30 minutes.

  By repeating the above steps (1) and (2), it is possible to quickly and easily pattern a plurality of different lipid membrane-containing materials on the base material. After the completion of step (2), before performing step (1) again, a washing step for removing the unreacted lipid membrane-containing material from the substrate may be performed.

  In one embodiment, the patterning method of the present invention includes the following steps: (3) washing the substrate obtained in step (2) to remove unreacted lipid membrane-containing materials from the substrate; 4) Form a plurality of different lipid membrane-containing patterns by repeating steps (1) to (3) using a lipid membrane-containing material different from the lipid membrane-containing material used in the previous step. , May further be included.

  One aspect of the present invention is a method for isolating a lipid membrane-containing material, comprising the following steps: (1) “substrate of the present invention” or “substrate having the group of formula (III) of the present invention” (2) detecting the position of the lipid film-containing material on the substrate based on microscopic observation; (3) irradiating the position with light, and The lipid membrane-binding group of is allowed to bind to the lipid membrane, and the lipid membrane-containing material is immobilized on the substrate; (4) The substrate obtained in step (3) is washed and immobilized on the substrate. The method comprising removing unmodified material from the substrate. (Hereinafter also referred to as “the isolation method of the present invention”).

  The lipid membrane-containing material to be isolated by the isolation method of the present invention is not particularly limited, and examples thereof include circulating tumor cells (Circulating Tumor Cells: CTC).

  The detection based on the microscopic observation of the lipid membrane-containing material to be isolated by the isolation method of the present invention is based on the specific form and characteristics of the lipid membrane-containing material to be isolated without labeling the lipid membrane-containing material. It may be done. Moreover, the lipid membrane-containing material to be isolated may be labeled and performed based on the label.

  The light irradiation conditions in step (2) and the immobilization conditions for the lipid membrane-containing material in the isolation method of the present invention can be performed under the same conditions as in steps (1) and (2) of the patterning method of the present invention.

  The present invention will be described in more detail with reference to the following Examples and Comparative Examples, but it goes without saying that the scope of the present invention is not limited by these Examples.

In addition, the symbol used in the following Example shows the following meaning.
1H-NMR: Proton nuclear magnetic resonance spectrum
APTES: 3-Aminopropyltriethoxysilane
BOC: N-tert-butoxycarbonyl
BSA: Bovine serum albumin
CDCl ₃ : Deuterated chloroform
CHCl ₃ : Chloroform
DCC: N, N'-dicyclohexylcarbodiimide
DCM: dichloromethane
DMEM: Dulbecco's Modified Eagle Medium
DMF: N, N-dimethylformamide
DMSO: Dimethyl sulfoxide
eq: chemical equivalent
Et ₃ N: Triethylamine
FBS: Fetal bovine serum
MeOH: methanol
MW: molecular weight
NHS: N-hydroxysuccinimide
PBS: phosphate buffered saline
PEG: Polyethylene glycol
Ph: Phase contrast microscope
PL: Photo cleavable linker, 4- [4- (1-hydroxy-ethyl) -2-methoxy-5-nitrophenoxy] butyric acid
Rf: retention factor
rpm: times per minute
TFA: trifluoroacetic acid
THF: tetrahydrofuran
TLC: Thin layer chromatography
WSC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

Example 1
Preparation of compounds of formula (Ic)
[Wherein n is 72. ]

(1) Production of compound 1c

In a 50 mL eggplant flask, oleylamine (MW: 267.5, 264.0 mg, 987.0 μmol, 2.01 eq) and 4- [4- (1-hydroxy-ethyl) -2-methoxy-5-nitrophenoxy] butyric acid (hereinafter “Photo (also called “cleavable linker” or “PL”) (MW: 299.28, 143.8 mg, 480.5 μmol, 1.0 eq) was added, and 5 mL of dry THF was added and stirred. WSC (MW: 191.7, 268.7 mg, 1401.6 μmol, 2.92 eq), NHS (MW: 115.1, 97.3 mg, 1.76 eq) and 3 drops of dry Et ₃ N were added and reacted for 17 hours. Completion of the reaction was confirmed by disappearance of the spot of PL (Rf: 0.4) by TLC (CHCl ₃ : MeOH = 6: 1). After drying under reduced pressure, silica gel was packed in a column tube having a diameter of 3.0 cm to a height of 10 cm, and the reaction product was loaded with 3 mL of CHCl ₃ to purify the column. By drying under reduced pressure, an oily yellow solid was obtained. Analysis was performed by 1H-NMR.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.40 (br m, CH3CH2, CH = CHCH2CH2CH2CH2CH2CH2, 22H); 1.47 (tt, CH2CH2NH, 2H); 1.56 (d, CH (OH) CH3, 3H); 1.93-2.08 (m, CH2CH = CH, 4H); 2.20 (tt, OCH2CH2, 2H); 2.40 (t, C (= O) CH2, 2H); 3.23 (dt, NHCH2 , 2H); 3.98 (s, OCH3, 3H); 4.12 (t, OCH2, 2H); 5.29-5.43 (br m, CH = CH, 2H); 5.58 (m, CHOH, 1H); 7.30 (s, aromatic (CH-m-NO ₂ , 1H); 7.58 (s, aromatic CH-O-NO ₂ , 1H).

(2) Production of compound 2c
Compound 1 (MW: 548.77, 261.0 mg, 475.6 μmol, 1 eq) was placed in a 50 mL eggplant flask and dried under reduced pressure. 2 mL of dry DCM was added and stirred. Add dry Et ₃ N (MW: 101.19, d = 0.726, 200 μL, 145.2 mg, 1.435 mmol, 3.0 eq), 4-nitrophenol chloroformate (MW: 201.56, 115.0 mg, 570.7 μmol, 1.2 eq) and add 30 After confirmation by TLC (CHCl 3: MeOH = 24: 1), the compound 1 spot (Rf: 0.33) almost disappeared, confirming the completion of the reaction. Silica gel was packed in a column tube having a diameter of 3.0 cm to a height of 15 cm, and the reaction product was loaded with 2.5 mL of CHCl ₃ to purify the column. Drying under reduced pressure and azeotropy with ethyl acetate gave a solid-wax yellow substance. Analysis was performed by 1H-NMR.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.40 (br m, CH3CH2, CH = CHCH2CH2CH2CH2CH2CH2, 22H); 1.48 (tt, CH2CH2NH, 2H); 1.78 (d, CH (O) CH3, 3H); 1.93-2.08 (m, CH2CH = CH, 4H); 2.21 (tt, OCH2CH2, 2H); 2.39 (t, C (= O) CH2, 2H); 3.25 (dt, NHCH2 , 2H); 4.00 (s, OCH3,3H); 4.14 (t, OCH2, 2H); 5.30-5.42 (br m, CH = CH, 2H); 6.54 (q, CH3CHO, 1H); 7.11 (s, aromatic CH-m-NO ₂ , 1H); 7.35 (d, aromatic CH-o-NO ₂ , 2H); 7.62 (s, aromatic CH-o-NO ₂ , 1H); 8.26 (d, aromatic CH-m-NO ₂ , 2H).

(3) Production of compound 3c
Place NHS oleate (MW: 379.53, 104.0 mg, 274.0 μmol, 1.5 eq) and mono-Boc-Lys-OH (MW: 246.3, 45.0 mg, 182.7 μmol, 1.0 eq) in a 50 mL eggplant flask and dry under reduced pressure. 2 mL of dry DMF was added. When the reaction was carried out for 30.5 hours and confirmed by TLC (CHCl ₃ : MeOH = 3: 1), mono-Boc-Lys-OH (Rf: 0.0) was not detected by ninhydrin, so the reaction was completed. After removing the solvent by vacuum decompression, the eggplant flask was azeotroped with MeOH under freezing with dry ice / MeOH. After dissolving in 4 mL of hexane + 4 mL of ethyl acetate, it was washed twice with 8 mL of MilliQ water using a separatory funnel. The organic layer was dried under reduced pressure, and a flash column was performed using a 1.5 cm diameter column tube and CHCl ₃ : MeOH = 12: 1 as a developing solvent. In TLC (CHCl ₃ : MeOH = 6: 1), a compound having a spot of Rf: 0.4 that appeared by staining with ninhydrin and heating with a lighter was collected and dried under reduced pressure. A liquid transparent compound was obtained, and 1H-NMR was measured with a CDCl ₃ solvent.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.40 (br m, CH3CH2, CH = CHCH2CH2CH2CH2CH2CH2, 22H); 1.42 (m, NHCH2CH2CH2, 2H); 1.45 (s, C (CH3) 3, 9H); 1.54 (m, NHCH2CH2, 2H); 1.71 (m, NHCHCH2, 2H); 1.87 (m, CH2CH2C (= O), 2H); 1.93-2.08 (m, CH2CH = CH, 4H); 2.17 (t, CH2C (= O), 2H); 3.25 (dt, NHCH2, 2H); 3.73 (dt, NHCHC (= O), 1H); 5.20-5.40 (br m, CH = CH, 2H ).

(4) Production of compound 4c
Compound 3c (MW: 510.75, 95.5 mg, 187.8 μmol, 1.0 eq) and NHS (MW: 115.1, 34.6 mg, 300.6 μmol, 1.6 eq) are placed in a 50 mL eggplant flask, and after drying under reduced pressure, 2 mL of dry DCM is added. It was. WSC (MW: 191.7, 68.5 mg, 357.3 μmol, 1.9 eq) was added and reacted for 1 hour. As confirmed by TLC (CHCl ₃ : MeOH = 12: 1), the disappearance of compound 3 (Rf: 0.25) and the appearance of the target product (Rf: 0.45) were confirmed by staining with ninhydrin and heating with a lighter. finished. The reaction solution was placed in a separatory funnel, washed with 8 mL of DCM, and washed once with 10 mL of MilliQ water. When the DCM layer was dried under reduced pressure, a transparent viscous liquid was obtained. 1H-NMR was measured with CDCl ₃ solvent.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.40 (br m, CH3CH2CH2, CH = CHCH2CH2CH2CH2CH2, 20H); 1.46 (s, C (CH3) 3, 9H); 1.52-1.72 (br m, NHCH2CH2CH2CH2, 6H); 1.91 (m, CH2CH2C (= O), 2H); 2.01 (m, CH2CH = CH, 4H); 2.85 (s, C (= O) CH2CH2C (= O) , 4H); 3.19-3.35 (m, NHCH2, 2H); 3.72 (m, NHCHC (= O), 1H); 5.34 (m, CH = CH, 2H).

(5) Production of compound 5c
Compound 4c (MW: 607.82, 40.0 mg, 65.8 μmol, 1.2 eq) and Cl ^- NH ₃ ⁺ -PEG-COOH (MW: 3400, 186.2 mg, 54.8 μmol, 1.0 eq) are placed in a 50 mL eggplant flask and dried under reduced pressure. After that, 2 mL of dry DCM was added. Dry Et ₃ N (MW: 101.19, d = 0.726, 40 μL, 29.0 mg, 287.0 μmol, 5.2 eq) was added to start the reaction. After 17 hours, disappearance of the spot of Cl ^— NH ₃ ⁺ -PEG-COOH (Rf: 0.34 broad) was confirmed by TLC (CHCl ₃ : MeOH = 3: 1) and ninhydrin, and dried under reduced pressure. The reaction was dissolved in 2.0 mL of DCM in 40 mL of stirring diethyl ether and added dropwise. After centrifuging at 10,000 rpm for 15 minutes, the supernatant was removed by decantation and then dried under reduced pressure to obtain a pale yellow solid. Analysis was performed by 1H-NMR.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.35 (br m, CH3CH2CH2, CH = CHCH2CH2CH2CH2CH2, 20H); 1.38-1.40 (br m, CH2CH2C (= O), NHCH2CH2CH2O, 4H); 1.44 (s, C (CH3) 3, 9H); 1.57-1.69 (br m, CH2CH2CH2C (= O) OH, 4H); 1.73-1.86 (br m, CH2C (= O) NHCH2CH2, 4H 2.01 (m, CH2CH = CH, 4H); 2.15 (t, CH2CHC (= O) NH, 2H); 2.32 (t, CH2C (= O) OH, 2H); 3.23 (m, CH2C (= O) NHCH2, 2H); 3.33 (s, CHC (= O) NHCH2, 2H); 3.47 (t, CHC (= O) NH, 1H); 3.50-3.80 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 5.34 (m, CH = CH, 2H).

(6) Production of compound 6c
Compound 5c (MW: 3856.27, 163.2 mg, 42.3 μmol, 1.0 eq) was placed in a 50 mL eggplant flask, and after drying under reduced pressure, 4.9 mL of dry DCM was added. 0.1 mL of TFA was added, reacted for 1 hour, 0.4 mL of TFA was added and reacted for 1 hour, and further reacted at 50 ° C. for 15 minutes. The resultant was dried under reduced pressure, dissolved in 4 mL of dry DCM, charged with 2 mL of TFA, and further reacted for 2 hours. The mixture was dried overnight under reduced pressure, dissolved in 40 mL of stirring diethyl ether and added dropwise to 1.5 mL of DCM. After centrifugation at 12000 rpm for 15 minutes, the supernatant was removed by decantation and dried under reduced pressure to obtain a light yellow solid.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.38 (br m, CH3CH2CH2, CH = CHCH2CH2CH2CH2CH2, 20H); 1.38-1.48 (br m, CH2CH2C (= O), NHCH2CH2CH2O, 4H); 1.53-1.72 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.73-1.86 (br m, CH2C (= O) NHCH2CH2, 4H); 2.00 (m, CH2CH = CH, 4H); 2.29 (t, CH2CHC (= O) NH, 2H); 2.34 (t, CH2C (= O) OH, 2H); 3.27 (m, CH2C (= O) NHCH2, 2H); 3.33 (s , CHC (= O) NHCH2, 2H); 3.48 (m, CHC (= O) NH, 1H); 3.50-3.80 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 5.34 (m, CH = CH, 2H ).

(7) Production of compound 7c
In a 50 mL eggplant flask, compound 6c (MW: 3870.18, 87.1 mg, 22.5 μmol, 1.0 eq) and compound 2c (MW: 713.39, 35.2 mg, 49.3 μmol, 2.19 eq) are placed. After drying under reduced pressure, 2 mL of dry DCM is added. I put it in. Dry Et ₃ N (MW: 101.19, d = 0.726, 10 μL, 29.1 mg, 287.6 μmol, 12.8 eq) was added and reacted for 41 hours. When confirmed with TLC (CHCl ₃ : MeOH = 6: 1) and ninhydrin, no coloration occurred, and the reaction was completed. To 40 mL of stirring diethyl ether, dissolved in 1.5 mL of DCM was added dropwise. After centrifugation at 12000 rpm for 15 minutes, the supernatant was removed by decantation and dried under reduced pressure to obtain a light yellow solid.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.36 (br m, CH3CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, 42H); 1.38-1.53 (br m, CH2CH2CH2CH2C (= O ) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 4H); 2.00 (m, CH2CH = CH, 8H); 2.08-2.24 (br m, CH2CHC (= O) NH, NHC (= O) CH2CH2CH2O, 4H); 2.32 (t, CH2C (= O) OH, 2H); 2.39 (m, CH2CH2NHC (= O) CH2, 4H); 3.24 (m, CH2C (= O) NHCH2, 2H); 3.33 (m, CHC (= O) NHCH2, 2H); 3.48 (m, CHC (= O) NH, 1H) ; 3.50-3.80 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 3.97 (d, OCH3, 3H); 4.10 (t, NHC (= O) CH2CH2CH2O, 2H); 5.34 (m, CH = CH, 2H) ; 5.62 (m, CHC (= O) NH, 1H); 6.32 (m, CHO, 1H); 7.08 (s, aromatic CH-m-NO ₂ , 1H); 7.55 (s, aromatic CH-o-NO ₂ , 1H).

(8) Production of compound of formula (Ic)
Compound 7c (MW: 4331.35, 86.5 mg, 20.0 μmol, 1.0 eq), NHS (MW: 115.1, 4.6 mg, 40.0 μmol, 2.0 eq), DCC (MW: 206.33, 8.5 mg, 41.2 μmol, in a 50 mL eggplant flask 2.1 eq) was added, and after drying under reduced pressure, 2 mL of dry DCM was added. The mixture was reacted for 3 hours and dried under reduced pressure. The solution was added dropwise to 1.3 mL of DCM dissolved in 40 mL of stirring diethyl ether. After centrifugation at 12000 rpm for 15 minutes, the supernatant was removed by decantation and dried under reduced pressure to obtain a pale yellow solid.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.36 (br m, CH3CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, 42H); 1.41-1.54 (br m, CH2CH2CH2CH2C (= O ) NH, NHCH2CH2CH2O, 4H); 1.53-1.69 (br m, CH2CH2CH2C (= O) O, CH2CH2CH (NH2), 6H); 1.70-1.83 (m, CH2C (= O) NH, 4H); 2.01 (m, CH2CH = CH, 8H); 2.08-2.24 (br m, CH2CHC (= O) NH, NHC (= O) CH2CH2CH2O, 4H); 2.38 (m, CH2CH2NHC (= O) CH2, 4H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24 (m, CH2C (= O) NHCH2, 2H); 3.34 (m, CHC (= O) NHCH2, 2H); 3.47 (m, CHC (= O) NH, 1H); 3.50-3.80 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 3.96 (d, OCH3, 3H); 4.10 (m, NHC ( = O) CH2CH2CH2O, 2H); 5.34 (m, CH = CH, 2H); 5.62 (m, CHC (= O) NH, 1H); 6.35 (m, CHO, 1H); 7.03 (s, aromatic CH-m -NO ₂ , 1H); 7.57 (s, aromatic CH-o-NO ₂ , 1H).

Example 2: Manufacturing method of substrate for immobilizing cells (1) Preparation of collagen-coated substrate A slide glass was placed in a 4-well plate, and 4 mL of 0.3 mg / mL collagen / pH 3.0 HCl solution was added. It was allowed to stand at room temperature overnight, washed 3 times with MilliQ water and dried.
(2) Production of Substrate for Immobilizing Lipid Membrane-Containing Substance A solution obtained by diluting a DMSO solution (1 mM) of the compound of formula (Ic) produced in Example 1 with a PBS solution 100 times as described above ( The collagen-coated substrate prepared in 1) was reacted in a dish having a diameter of 3.5 cm for 4 hours at 37 ° C. The PBS solution of the compound of the formula (Ic) produced in Example 1 was removed, washed with 1 mL of MilliQ water 6 times, and then dried to produce a substrate for immobilizing cells.

Example 3 Fabrication of High Density Single Cell Array Contact exposure (0.6 mW / cm ² , 3000 seconds, 1.8 J / cm ^{2) to} the surface of the substrate prepared in Example 2 using the photomask shown in FIG. And installed on the bottom of a 3.5 cm dish. Put 2 mL of PBS solution here, seed with 1 mL of 5 × 10 ⁶ cells / mL Ba / F3 cell / PBS suspension, let stand for 15 minutes, and wash the unfixed cells with PBS solution. . Ba / F3 cells were stained with 1 mL of 0.25 μg / mL calcein-AM / PBS for 15 minutes and then observed with a microscope (fluorescence, bright field, × 4 objective lens). The results are shown in FIG.

A very dense single cell array could be made. One cell was fixed in a 14 μm square, and the cell density was 5.1 × 10 ⁵ cells / cm ² .

Example 4: Patterning of multiple types of cells in single cell units A dish with a diameter of 25 mm was prepared, the bottom of which was made of food wrap (film thickness: about 10 μm, polyvinylidene chloride). To this dish, 1 mL of 0.3 mg / mL Type I collagen / pH 3.0 HCl was added, and the collagen was coated overnight. Washed 3 times with MilliQ water and dried. A DMSO solution (1 mM) of the compound of formula (Ic) produced in Example 1 was reacted with a solution (1 mL) diluted 100-fold with a PBS solution for 4 hours at 37 ° C. Washed 6 times with MilliQ water and dried. A photomask was attached to the back of the bottom of the dish with a small amount of MilliQ water, and proximity exposure of 1.8 J / cm ² (365 nm, 1.0 mW / cm ² , 1800 seconds) was performed. 1 mL of PBS solution was added thereto, and 5 × 10 ⁶ cells / mL Ba / F3 (EGFP) cells / PBS 1 mL was seeded. After standing for 15 minutes, it was washed 6 times with 1 mL of PBS solution. Sticking a photomask on the back of the dish bottom, Puromiki in alignment is performed of a cell pattern and a photomask fabricated while observing with a microscope, again ^{1.8 J / cm 2 (365 nm} , 1.0 mW / cm 2, 1800 sec) City exposure was performed. Here, 1 mL of PBS suspension (5 × 10 ⁶ cells / mL) of Ba / F3 cells (Ba / F3 (CytoRed)) stained with CytoRed was seeded. After standing for 15 minutes, it was washed 6 times with 1 mL of PBS. A schematic diagram of the procedure of this experiment is shown in FIG. The cell pattern of the prepared multiple cell population was observed with a confocal microscope. The results are shown in FIG.

Example 5: Patterning of multiple cell populations by laser drawing using a confocal microscope 1 microchannel (μ-Slide VI ^0.1 , channel width 1 mm x total length 17 mm x height 0.1 mm, manufactured by ibidi) A / v% BSA / PBS solution was introduced and allowed to stand overnight at room temperature. Thereafter, 100 μL of MilliQ water was added to the chambers at both outlets of the flow path, and washing for removing was performed three times. 100 μL of MilliQ water was put into only one chamber, and the inside of the flow channel was washed by allowing the other chamber to flow through the flow channel at atmospheric pressure. The cleaning solution accumulated in both chambers was removed, and a solution obtained by diluting the compound of the formula (Ic) prepared in Example 1 in Example 1 with a DMSO solution (10 mM) 100 times with a PBS solution was added to only one chamber. By adding μL, the solution of the compound of formula (Ic) was caused to flow into the flow path by atmospheric pressure. By reacting at 37 ° C. for 4 hours, the compound of the formula (Ic) was bound to the substrate. 100 μL of MilliQ water was put in the chambers at both outlets of the flow path, and washing was performed 6 times for removal. 100 μL of MilliQ water was put into only one chamber, and the inside of the flow channel was washed by allowing the other chamber to flow through the flow channel at atmospheric pressure. A large chamber with a 5 mL syringe was added to the inlet of this flow path, and a tube connected to a syringe pump was attached to the outlet (FIG. 5).

As cells to be patterned, Ba / F3 cells showing different colors of fluorescence were prepared. As the green cells, Ba / F3 (EGFP) cells in which enhanced green fluorescent protein (EGFP) was expressed were used. As red cells, Ba / F3 (KO) cells in which Kusabira Orange was expressed were used. Ba / F3 (Hoechst) cells stained with Hoechst33342 were used as blue cells. As yellow cells, Ba / F3 (KO + Calcein) obtained by staining Ba / F3 (KO) cells with Calcein-AM (Dojindo Laboratories, Kumamoto, Japan) was used. As the light blue cells, Hoechst33342 and Ba / F3 (Hoschst + Calcein) stained with Calcein-AM were used. Each cell was suspended in PBS at a concentration of 1 × 10 ⁸ cells / mL to prepare a cell solution for patterning.

Each of the five types of cells was patterned into an arbitrary shape by repeating drawing with a laser beam, seeding of cells, and washing. The detailed method is as follows.
(1) Using a confocal microscope, a 405 nm laser was irradiated in an arbitrary shape on the bottom surface of the flow path on which the compound-modified surface of formula (Ic) was prepared.
(2) 50 μL of various cell solutions were placed in a large chamber installed at the channel inlet, and 20 μL of the solution was drawn with a syringe pump to introduce cells into the channel.
(3) After standing for 20 minutes, PBS was placed in a large chamber as a washing solution for cells not immobilized. Using a syringe pump, the flow path was washed with a PBS solution for 1 minute at a flow rate of 30 mL / h and then for 2 minutes at a flow rate of 60 mL / h.
This (1) laser drawing, (2) cell seeding, and (3) washing were performed in the order of green, yellow, light blue, blue, and red cells to pattern a plurality of cell populations.

  As a result, by using the compound-modified surface of the formula (Ic), it was possible to pattern a plurality of types of cell samples into arbitrary shapes. (FIGS. 6 and 7). In this experiment, 5 types of cells could be patterned in about 3 hours.

Example 6: Cell culture on substrate surface modified with compound (Ic) 500 μL of 0.3 mg / mL collagen / pH 3.0 HCl solution was applied onto a cover glass having a diameter of 34 mm and spin coater (20 seconds) Accelerated to 3000 rpm, rotated at 3000 rpm for 20 seconds, and stopped for 20 seconds). Washed 3 times with MilliQ water and dried. The collagen-coated substrate was placed in a 35 mm diameter dish, and reacted with 1.5 mL of a DMSO solution (1 mM) of compound (Ic) diluted 100-fold with a PBS solution at 37 ° C. for 3 hours. The PBS solution of the compound (Ic) was removed, washed 6 times with MilliQ water, and then dried to manufacture a substrate for immobilizing cells. This was placed on the bottom of a 6-well plate, sterilized with 1 mL of 70% ethanol, and washed twice with 2 mL of PBS. HEK293T cells (adherent cells, human embryonic kidney epithelial cell line) suspended in FBS-free DMEM medium were seeded at 2 × 10 ⁵ cells / well. After leaving still for 30 minutes, FBS was added to the medium so as to be 10% v / v. The cells were cultured in a 37 ° C, 5% CO ₂ environment for 3 days. After collecting cells by trypsin treatment, dead cells were stained with trypan blue, and the number of viable cells and cell viability were measured.

Regardless of light irradiation (365 nm, 1.6 J / cm ² ) or non-irradiation, the number of cells and cell viability in the culture on the surface of compound (Ic) are both positive, such as collagen surface and cell culture dishes. It was the same as the control (FIG. 8). From these results, it was shown that photofixed cells can be cultured on the surface of the compound (Ic) and have high cytocompatibility.

Example 7: One cell level patterning of adherent cells (HepG2 cells), primary cultured cells (NHDF cells), liposomes (rhodamine fluorescence) A cover glass having a diameter of 34 mm was immersed in a 0.3 mg / mL collagen / pH 3.0 HCl solution. Thereafter, spin coating (acceleration to 3000 rpm in 20 seconds, rotation at 3000 rpm for 20 seconds, and stopping for 20 seconds) was performed to coat collagen. Washed 3 times with MilliQ water and dried. A collagen-coated substrate was placed in a 35 mm diameter dish, and reacted with 1.5 mL of a DMSO solution of compound (Ic) (1 mM) diluted 100-fold with PBS solution at 37 ° C. for 4 hours. The PBS solution of compound (I-c) was removed, and washing with MilliQ water was performed 6 times, followed by drying to produce a substrate for immobilizing cells. This was placed on a photomask on which an array pattern was drawn, and contact exposure was performed at a wavelength of 365 nm and 1.6 J / cm ² . After sterilizing with 70% ethanol, it was washed 3 times with PBS.

Place this substrate in a 35 mm diameter dish, add 2 mL of PBS, then HepG2 cells (human hepatoma-derived cell line, adherent cells) or NHDF cells (normal human skin fibroblasts, primary cultured cells, adhesiveness) 1 mL of PBS suspension of cells) (3 × 10 ⁶ cells / ml) was added, and after 5 minutes, non-immobilized cells were washed away. In the fixed HepG2 cells, the medium was replaced with DMEM medium containing 10% v / v FBS and cultured at 37 ° C for 2 hours. The HepG2 cells that were arrayed in a single cell were stretched and adhered to the substrate. Was confirmed (FIG. 9). Even in the immobilized NHDF cells, the medium was replaced with 10% v / v FBS-containing DMEM medium and cultured at 37 ° C for 15 minutes. As a result, NHDF cells that were arrayed in one cell were stretched and adhered to the substrate. This was confirmed (FIG. 10).

  On the same substrate as above, after contact exposure of the array pattern, 1 mL of PBS (F60203F-R, FormuMax Scientific) solution for PBS 100-fold dilution was added and washed 10 minutes later, and the liposome array pattern was confirmed. (FIG. 11).

Example 8: Patterning of quantum dots The surface of the compound (Ic) -modified microchannel prepared by the same method as in Example 5 was irradiated with a human-type pattern using 405 nm laser light. Biotin-modified lipid-coated quantum dots (biotin-labeled Qdot (registered trademark) 655 nanocrystals, manufactured by Thermo Fisher Scientific) were diluted to 20 nM with Qdot (registered trademark) incubation buffer (produced by Thermo Fisher Scientific). And left to stand for 5 minutes. When quantum dots that were not immobilized with PBS were washed away, it was confirmed that the quantum dots were patterned in a human mold (FIG. 12). In addition, we tried patterning a 10 μg / mL MilliQ aqueous solution of carboxylic acid-modified lipid-coated quantum dots (CdSeS / ZnS alloyed quantum dots, COOH functionalized, fluorescence λem 525 nm, manufactured by Sigma-Aldrich) by the same method. It was confirmed that quantum dots were patterned on the mold (FIG. 13).

Example 9: Co-patterning of non-adherent and adherent cells A 34 mm diameter cover glass was immersed in a 0.3 mg / mL collagen / pH 3.0 HCl solution followed by spin coating (accelerated to 3000 rpm in 20 seconds, 3000 rpm The coating of collagen was performed by rotating for 20 seconds and stopping for 20 seconds). Washed 3 times with MilliQ water and dried. The collagen-coated substrate was placed in a 35 mm diameter dish, and reacted with 1.5 mL of a solution of compound (Ic) in DMSO (1 mM) diluted 100-fold with PBS solution at 37 ° C. for 4 hours. The PBS solution of the compound (I-c) was removed, and washing with MilliQ water was performed 6 times, followed by drying to produce a substrate for immobilizing cells. This was placed on a photomask on which a line pattern (width 200 μm, interval 200 μm) was drawn, and contact exposure was performed at a wavelength of 365 nm and 1.6 J / cm ² . After sterilizing with 70% ethanol, it was washed 3 times with PBS. Place this substrate in a 35 mm diameter dish, add 2 mL of PBS, and then add 1 mL of PBS suspension (5 × 10 ⁶ cells / ml) of Ba / F3 (KO) cells expressing red fluorescent protein. Add mL. After standing for 10 minutes, the non-immobilized cells were washed away with PBS and placed in a dish (WillCo-Dish, WillCo Wells) with a diameter of 50 mm and a glass observation window diameter of 2 mL of PBS. . This is placed on a photomask on which a line pattern (width 200 μm, interval 200 μm) is drawn so that the line pattern of the Ba / F3 (KO) cells and the line pattern of the photomask are orthogonal, and the wavelength is 365 nm, 1.6 Exposure was performed at J / cm ² . Transfer the cell patterning substrate into a 35 mm diameter dish, add 2 mL of PBS, and then add 1 mL of PBS suspension (2.5 × 10 ⁶ cells / ml) of HEK293T (EGFP) cells expressing green fluorescent protein. I put it in. After standing for 10 minutes, the non-immobilized cells were washed and removed with PBS, and two orthogonal line patterns composed of Ba / F3 (KO) cells and HEK293T (EGFP) cells were confirmed (left side of FIG. 14). ). Further, when the medium was replaced with a DMEM medium containing 10% v / v FBS and cultured at 37 ° C. for 3 hours, it was confirmed that HEK293T (EGFP) cells were stretched and adhered to the substrate (FIG. 14 right). .

  In addition, line patterning of liposomes (rhodamine fluorescence) followed by line patterning of HEK293T (EGFP) cells was performed in the same manner, and two orthogonal configurations composed of liposomes (rhodamine fluorescence) and HEK293T (EGFP) cells, respectively. One line pattern was confirmed (FIG. 15). At this time, a photomask on which a line pattern having a width of 100 μm and an interval of 100 μm was drawn was used.

Example 10: CTC sorting from tumor bearing mice
As a model experiment for CTC isolation, light capture of cancer cells from the peripheral blood of tumor-bearing mice was performed. Peripheral blood was collected from the heart of mice bearing DLD1 cells. Peripheral blood was treated with 0.9% ammonium chloride to lyse erythrocytes, and then centrifuged and washed with PBS. After reacting with 100 nM biotin-modified anti-Epiregulin antibody for 15 minutes, 10 nM streptavidin-modified quantum dots (Qdot (registered trademark) 655 Streptavidin Conjugate, Thermo Fisher Scientific) are reacted for 5 minutes. Cancer cells were fluorescently labeled. This cell solution was introduced into the microchannel coated with compound (Ic) in the same manner as in Example 5, and cancer cells were detected from the fluorescence of the quantum dots (ex / em 488/655 nm). By irradiating only the detected cancer cells with a 405 nm laser, the cancer cells were fixed in the flow path, and other cells were washed away with PBS. As a result, it succeeded in selectively isolating cancer cells from the peripheral blood into the channel (FIG. 16).

Example 11: Synthesis of compound (II-b) which is a photoactivated PEG lipid derivative (1) Synthesis of compound 1d
Put 20 mL of ethanol and 10 mL of MilliQ water in a 100 mL eggplant flask, and add 4- (4-tert-butoxycarbonylpiperazinyl) phenylboronic acid pinacol ester (MW: 388.32, 154.0 mg, 397 μmol, 1.0 eq), 1- (5-bromo-2-nitrophenyl) ethanone (MW: 244.04, 127.3 mg, 522 μmol, 1.3 eq), potassium carbonate (MW: 138.2, 158.3 mg, 1.15 mmol, 2.9 eq), tetrabutyl Ammonium bromide (MW: 322.4, 126.2 mg, 391 μmol, 1.0 eq) was added, and a catalytic amount of palladium (II) acetate was added. The reflux reaction was performed in a microwave reactor at 150 ° C. for 10 minutes at 150 W output. After allowing to cool, 40 mL of MilliQ water was added, and extraction was performed twice with 40 mL of ethyl acetate. The extraction solution was washed twice with 40 mL of saturated saline, and further washed twice with 40 mL of MilliQ water. After drying the organic layer under reduced pressure, a 3.0 cm diameter column tube was filled with silica gel to a height of 5 cm, and the crude product was loaded with 4.0 mL of a solvent of hexane: ethyl acetate = 1: 1, and hexane: ethyl acetate = 5. 1: followed by column purification with a developing solvent of hexane: ethyl acetate = 3: 1. When dried under reduced pressure, 109.0 mg (yield 65%) of an orange solid was obtained.
¹ H NMR (600 MHz, CDCl ₃ ) δ 1.49 (s, C (CH3) 3, 9H); 2.58 (s, C (= O) CH3, 3H); 3.26 (br, C (= O) N ( (CH) 2, 2H); 3.61 (br, N (CH) 2, 2H); 7.00 (d, aromatic NC (CH) 2, 2H); 7.51 (s, aromatic C (= O) CCH, 1H); 7.56 (d, aromatic C (CH) 2, 2H); 7.71 (d, aromatic CH-m-NO ₂ , 1H); 8.17 (d, aromatic CH-o-NO ₂ , 1H)

(2) Synthesis of compound 2d
Compound 1d (MW: 425.48, 109.0 mg, 256 μmol, 1.0 eq) and sodium borohydride (MW: 37.83, 14.8 mg, 391 μmol, 1.5 eq) were placed in a 50 mL eggplant flask and dried under reduced pressure. 5 mL dry MeOH and 5 mL dry THF were added on ice and stirred. After 2 hours, as confirmed by TLC (hexane: ethyl acetate = 3: 1), the compound 1d spot (Rf: 0.17) disappeared, confirming the completion of the reaction. After drying under reduced pressure, the residue was dissolved in 50 mL of ethyl acetate, washed twice with 50 mL of saturated brine, and further washed twice with 50 mL of MilliQ water. When dried under reduced pressure, 107.3 mg (yield 98%) of an orange solid was obtained.
¹ H NMR (600 MHz, CDCl ₃ ) δ 1.49 (s, C (CH3) 3, 9H); 1.63 (s, C (= O) CH3, 3H); 3.25 (br, C (= O) N ( (CH) 2, 2H); 3.61 (br, N (CH) 2, 2H); 5.55 (m, CHOH, 1H); 7.01 (d, aromatic NC (CH) 2, 2H); 7.58 (d, aromatic CH- m-NO ₂ , 1H); 7.59 (d, aromatic C (CH) 2, 2H); 8.02 (d, aromatic CH-o-NO ₂ , 1H); 8.03 (s, aromatic C (OH) CCH, 1H)

(3) Synthesis of compound 3d
Compound 2d (MW: 427.49, 107.3 mg, 251 μmol, 1.0 eq) was placed in a 50 mL eggplant flask and dried under reduced pressure. 10 mL of 4N HCl / ethyl acetate was added, and the mixture was stirred for 30 minutes and then dried under reduced pressure. 116.0 mg (yield> 99%) of an orange solid was obtained.
¹ H NMR (600 MHz, CDCl ₃ ) δ 1.60 (s, C (= O) CH3, 3H); 3.46 (br, NH (CH) 2, 2H); 3.66 (br, N (CH) 2, 2H 5.52 (m, CHOH, 1H); 7.16 (d, aromatic NC (CH) 2, 2H); 7.58 (d, aromatic C (CH) 2, 2H); 7.79 (d, aromatic CH-m-NO ₂ , 1H); 8.02 (d, aromatic CH-o-NO ₂ , 1H); 8.07 (s, aromatic C (OH) CCH, 1H)

(4) Synthesis of compound 4d
Compound 3d (MW: 363.84, 91.0 mg, 250 μmol, 1.0 eq) and NHS oleate (MW: 379.53, 142.0 mg, 374 μmol, 1.5 eq) were placed in a 50 mL eggplant flask and dried under reduced pressure. Dry DCM 5 mL and dry Et ₃ N (MW: 101.19, d = 0.726, 360 μL, 261.4 mg, 2.58 mmol, 10 eq) were added and stirred. After 1.5 hours, as confirmed by TLC (CHCl ₃ : MeOH = 6: 1), the spot of compound 3d (Rf: 0.2) disappeared, confirming the completion of the reaction. After drying under reduced pressure, the residue was dissolved in 50 mL of ethyl acetate, washed twice with 50 mL of saturated brine, and further washed twice with 50 mL of MilliQ water. After drying under reduced pressure, the suspension was suspended in 40 mL of cyclohexane, centrifuged at 10,000 G for 3 minutes at 10 ° C., and then washed to remove the supernatant three times. When dried under reduced pressure, 115.6 mg (yield 78%) of an orange solid was obtained.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, 20H); 1.60-1.72 (m, CH (OH) CH3, NC (= O) CH2CH2, 5H); 2.01 (m, CH2CH = CH, 4H); 2.38 (t, C (= O) CH2, 2H); 3.22-3.32 (br, N (CH) 2, 2H); 3.66,3.81 (br, C (= O) N (CH) 2, 2H); 5.35 (m, CH = CH, 2H); 5.55 (m, CHOH, 1H); 7.01 (d, aromatic NC (CH) 2 , 2H); 7.58 (d, aromatic CH-m-NO ₂ , 1H); 7.59 (d, aromatic C (CH) 2, 2H); 8.02 (d, aromatic CH-o-NO ₂ , 1H); 8.03 ( s, aromatic C (OH) CCH, 1H)

(5) Synthesis of compound 5d
Compound 4d (MW: 591.82, 115.6 mg, 195.3 μmol, 1.0 eq) and 4-nitrophenol chloroformate (MW: 201.56, 68.8 mg, 341 μmol, 1.7 eq) were placed in a 50 mL eggplant flask and dried under reduced pressure. Dry DCM 5 mL and dry Et ₃ N (MW: 101.19, d = 0.726, 500 μL, 363 mg, 3.59 mmol, 18 eq) were added and stirred. After 4 hours, as confirmed by TLC (hexane: ethyl acetate = 1: 1), the spot of compound 4d (Rf: 0.22) disappeared, confirming completion of the reaction. After drying under reduced pressure, a 2.0 cm diameter column tube was filled with silica gel to a height of 15 cm, and the reaction product was loaded with 3.0 mL of hexane: ethyl acetate 1: 1 solvent, and hexane: ethyl acetate = 3: 2 developing solvent. Column purification. When dried under reduced pressure, 140.2 mg (yield 95%) of an orange solid was obtained.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, 20H); 1.67 (t, NC (= O) CH2, 2H ); 1.84 (d, CH (OH) CH3, 3H); 2.01 (m, CH2CH = CH, 4H); 2.38 (t, C (= O) CH2, 2H); 3.24-3.33 (br, N (CH) 2,2H); 3.67,3.82 (br, C (= O) N (CH) 2, 2H); 5.35 (m, CH = CH, 2H); 6.52 (m, CHOH, 1H); 7.03 (d, aromatic NC (CH) 2, 2H); 7.34 (d, aromatic C (= O) OC (CH) 2, 2H); 7.58 (d, aromatic C (CH) 2, 2H); 7.65 (d, aromatic CH-m -NO ₂ , 1H); 7.87 (s, aromatic C (OH) CCH, 1H); 8.11 (d, aromatic CH-o-NO ₂ , 1H); 8.25 (d, aromatic (CH) 2-o-NO ₂ , 2H)

(6) Synthesis of compound 6d
Compound 6c (MW: 3870.18, 116.0 mg, 30.0 μmol, 1.0 eq) and compound 5d (MW: 756.93, 31.2 mg, 41.2 μmol, 1.4 eq) were placed in a 50 mL eggplant flask and dried under reduced pressure. Dry DCM 3 mL and dry Et ₃ N (MW: 101.19, d = 0.726, 42 μL, 30.5 mg, 301 μmol, 10 eq) were added and stirred. Two days later, TLC (CHCl ₃ : MeOH = 6: 1) and ninhydrin confirmed that no coloration occurred, so the reaction was completed. To 40 mL of stirring diethyl ether, dissolved in 1.5 mL of DCM was added dropwise. After centrifuging at 10,000 g for 10 minutes, the supernatant was removed by decantation and dried under reduced pressure to obtain 125.6 mg (yield 96%) of a pale yellow solid.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, 44H); 1.38-1.53 (br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 4H); 2.00 (m , CH2CH = CH, 8H); 2.08-2.24 (br m, CH2CHC (= O) NH, NHC (= O) CH2CH2CH2O, 4H); 2.32 (t, CH2C (= O) OH, 2H); 2.39 (m, CH2CH2NHC (= O) CH2, 4H); 3.24-3.40 (br m, N (CH) 2, CH2C (= O) NHCH2, CHC (= O) NHCH2, 6H); 3.48 (m, CHC (= O) NH , 1H); 3.50-3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2, C (= O) N (CH) 2); 5.34 (m, CH = CH, 4H); 5.59 (m, CHC (= O ) NH, 1H); 6.37 (m, CHO, 1H); 7.05 (d, aromatic NC (CH) 2, 2H); 7.56-7.62 (m, aromatic C (CH) 2, aromatic CH-m-NO ₂ , 3H); 7.82 (s, aromatic OCH (CH3) CCH, 1H); 8.05 (t, aromatic CH-o-NO 2, 1H);

(7) Synthesis of compound (II-b)
Compound 6d (MW: 4373.97, 21.9 mg, 5.01 μmol, 1.0 eq), NHS (MW: 115.1, 0.9 mg, 7.51 μmol, 1.5 eq), DCC (MW: 206.33, 2.1 mg, 10.2 μmol) in a 10 mL eggplant flask , 2.0 eq), and after drying under reduced pressure, 1 mL of dry DCM was added. The mixture was reacted for 21 hours and dried under reduced pressure. 0.8 mL of DCM was added and the precipitate was removed by filtration and added dropwise to 12 mL of stirring diethyl ether. After centrifuging at 10000 g for 10 minutes, the supernatant was removed and dried under reduced pressure. As a result, 21.7 mg (yield 98%) of a pale yellow solid was obtained.
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, 44H); 1.38-1.53 (br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 4H); 2.00 (m , CH2CH = CH, 8H); 2.08-2.24 (br m, CH2CHC (= O) NH, NHC (= O) CH2CH2CH2O, 4H); 2.39 (m, CH2CH2NHC (= O) CH2, 4H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, N (CH) 2, CH2C (= O) NHCH2, CHC ( = O) NHCH2, 6H); 3.48 (m, CHC (= O) NH, 1H); 3.50-3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2, CON (CH) 2); 5.34 (m, CH = CH, 4H); 5.59 (m, CHC (= O) NH, 1H); 6.37 (m, CHO, 1H); 7.05 (d, aromatic NC (CH) 2, 2H); 7.56-7.62 (m, aromatic C (CH) 2, aromatic CH-m-NO ₂ , 3H); 7.82 (s, aromatic OC (CH3) CCH, 1H); 8.05 (t, aromatic CH-o-NO ₂ , 1H)

Example 10: Evaluation of Cell Immobilization Function of Compound (II-b) In the same manner as in Example 5, the microchannel was coated with BSA and then the compound (II-b) was modified. However, a solution in which the compound (II-b) was dissolved in H ₂ O: DMSO = 1: 1 (v / v) was used for substrate modification. In addition, washing after modification with compound (II-b) was performed with PBS. Irradiate the microchannel with 405 nm light, and put 45 μL of Ba / F3 (EGFP) cells (2 × 10 ⁸ cells / mL) in PBS into only one chamber of the microchannel. The cells were caused to flow into the flow path. After standing for 30 minutes, non-immobilized cells were washed away with 1 mL of PBS, and the cells immobilized in the channel were observed with a fluorescence microscope. Although the cells were hardly immobilized on the surface not irradiated with light (FIG. 17 left), the cells were immobilized on the light irradiated surface (right of FIG. 17). Therefore, it was confirmed that compound (II-b) can also be used for cell immobilization by light irradiation.

Reference example 1
Examination about the effect of a lipid membrane binding inhibitory group The following model compounds (Ie) to (Ik) were synthesized, and a substituent functioning as a lipid membrane binding inhibitory group was examined.

(1) Synthesis of compounds (Ie, If, Ig, Ih, Ii, Ij) (1-1) Synthesis of compounds 1f, 1g, 1h, 1j
Lauric acid, pentadecylic acid, stearic acid and lignoceric acid were activated with NHS. These fatty acid NHS active esters were commonly synthesized by methods known to those skilled in the art shown below. Each fatty acid and an excess of N-hydroxysuccinimide were reacted in dry DCM using WSC as a condensing agent. After drying under reduced pressure, the residue was dissolved in ethyl acetate, followed by separation and washing twice with a saturated saline solution and then twice with water. The organic phase was dried under reduced pressure to obtain NHS active esters of various lipids as white solids.

(1-2) Synthesis of Compounds (Ie), (If), (Ig), (Ih), (Ii), (Ij) Fatty Acid NHS Activity Obtained by Synthesis Compounds 1f, 1g, 1h, 1j, which are esters, and purchased compounds 1e (N-acetoxysuccinimide) and compound 1i (oleic acid N-hydroxysuccinimide ester) are coupled to the amino group of compound 6c, followed by PEG-terminal carboxylic acid The acid was NHS active esterified. The reaction of these NHS active esters with amino groups, and the NHS active esterification of carboxylic acids were commonly performed by methods known to those skilled in the art shown below. Compound 6c and an excess of fatty acid NHS active ester were reacted in dry DCM in the presence of dry Et ₃ N. After confirming completion of the reaction using ninhydrin, vacuum drying was performed. Excess N-hydroxysuccinimide and DCC were added and reacted in dry DCM. By filtering the reaction solution, excess N-hydroxysuccinimide and DCC-derived reaction byproducts were removed. This was added dropwise to stirring diethyl ether, and after centrifugation, the supernatant was removed by decantation. When dried under reduced pressure, compounds (Ie), (If), (Ig), (Ih), (Ii), and (Ij) were obtained as white solids.

Compound (Ie)
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 3H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, 24H); 1.38-1.53 (br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 6H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 2H); 2.00-2.08 (m, CH2CH = CH, CH3C (= O) NH, 7H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50-3.88 (br m, CH2O (CH2CH2O) nCH2 , OCH2CH2CH2CH2); 4.34 (m, CHC (= O) NH, 1H); 5.34 (m, CH = CH, 2H)

Compound (If)
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, CH3 (CH2) 9, 44H); 1.38-1.53 ( br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 2H); 2.00 (m, CH2CH = CH, 4H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.21 (m, CH2C (= O) NHCH, 2H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50 -3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 4.34 (m, CHC (= O) NH, 1H); 5.34 (m, CH = CH, 2H)

Compound (Ig)
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, CH3 (CH2) 12, 50H); 1.38-1.53 ( br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 2H); 2.00 (m, CH2CH = CH, 4H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.21 (m, CH2C (= O) NHCH, 2H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50 -3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 4.34 (m, CHC (= O) NH, 1H); 5.34 (m, CH = CH, 2H)

Compound (Ih)
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, CH3 (CH2) 15, 56H); 1.38-1.53 ( br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 2H); 2.00 (m, CH2CH = CH, 4H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.21 (m, CH2C (= O) NHCH, 2H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50 -3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 4.34 (m, CHC (= O) NH, 1H); 5.34 (m, CH = CH, 2H)

Compound (I-i)
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, 44H); 1.38-1.53 (br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 2H); 2.00 (m , CH2CH = CH, 4H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.21 (m, CH2C (= O) NHCH, 2H); 2.62 (t, CH2C (= O) O, 2H) 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50-3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 4.34 (m, CHC (= O) NH, 1H); 5.34 (m, CH = CH, 4H)

Compound (Ij)
¹ H NMR (600 MHz, CDCl ₃ ) δ .0.88 (t, CH2CH3, 6H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, CH3 (CH2) 21, 68H); 1.38-1.53 ( br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) OH, CH2CH2CH (NH2), 6H); 1.70-1.84 (m, CH2C (= O) NH, 2H); 2.00 (m, CH2CH = CH, 4H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.21 (m, CH2C (= O) NHCH, 2H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50 -3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 4.34 (m, CHC (= O) NH, 1H); 5.34 (m, CH = CH, 2H)

(2) was a compound synthesized (2-1) Compound 1k Synthesis Example 1 (2) in the compounds c made in a similar manner with cholesterol p- nitrophenol derivative of the (I-k).

(2-2) Synthesis of Compound 2k Compound 1k was bound to compound 6c in the same manner as in the production of compound 7c in Example 1 (7).

(2-3) Synthesis of compound (Ik)
Compound (Ik) was obtained by a method similar to the method described in Example 1 (8).
¹ H NMR (600 MHz, CDCl ₃ ) δ. 0.68 (s, CH3C (CH) 2CH2, 3H), 0.84-0.93 (m, CH2CH3, (CH3) 2CH, CH3CH (CH) CH2, 12H); 1.00 (s , CH3C (CH) (CH2) C, 3H); 1.20-1.36 (br m, CH3CH2CH2CH2CH2 CH2CH2, CH = CHCH2CH2CH2CH2CH2, NHCH2CH2CH2CH2, CH3 (CH2) 21, 68H); 1.38-1.53 (br m, CH2CH2CH2CH2C (= O) NH, NHCH2CH2CH2O, 4H); 1.55-1.68 (br m, CH2CH2CH2C (= O) O, CH2CH2CH (NH2), 6H); 1.70-1.91 (m, CH2C (= O) NH, CCH2CH, 4H); 2.00 (m , CH2CH = CH, 4H); 2.15 (br m, NHC (= O) CH2CH2CH2O, 2H); 2.62 (t, CH2C (= O) O, 2H); 2.85 (s, C (= O) CH2CH2C (= O ), 4H); 3.24-3.40 (br m, CH2C (= O) NHCH2, CHC (= O) NHCH2, 4H); 3.50-3.88 (br m, CH2O (CH2CH2O) nCH2, OCH2CH2CH2CH2); 4.08 (br, CH (CH2) O, 1H); 4.34 (m, CHC (= O) NH, 1H); 4.47 (br, CH = C, 1H); 5.34 (m, CH = CH, 2H)

In the same manner as in Example 5, the microchannel was coated with BSA and subsequently modified with various PEG lipid derivatives. However, a solution in which PEG lipid was dissolved in H ₂ O: DMSO = 1: 1 (v / v) was used for substrate modification. Further, washing after PEG lipid modification was performed with PBS. 100 μL of Ba / F3 (EGFP) cell (1 × 10 ⁷ cells / mL) PBS suspension was placed in only one chamber of the microchannel, and the cells were allowed to flow into the channel by atmospheric pressure. After standing for 30 minutes, non-immobilized cells were washed away with 1 mL of PBS, and the number of cells immobilized in the flow channel was counted. The results are shown in FIG.

  Cells were immobilized in the flow channel modified with the compound (Ie), which is a model when the lipid membrane binding inhibitory group is a methyl group, and the cell immobilization function was not inhibited. On the other hand, compounds (I-f, I-g), which are models when the lipid membrane binding inhibitory group is a hydrophobic group such as an alkyl group (C11, C14, C17 and C23) longer than methyl and a cholesteryl group. , Ih, Ii, Ij, Ik), the cells were not immobilized, and the lipid membrane binding function was inhibited. Therefore, it was shown that when the lipid membrane binding inhibitory group is a hydrophobic group, the lipid membrane binding function is preferably inhibited.

  According to the present invention, not only a single type of lipid membrane-containing material but also a plurality of types of lipid membrane-containing materials can be patterned quickly and easily. Therefore, for example, a high-throughput drug screening is possible, for example, by arranging a plurality of different types of cells on the same substrate and examining a disease state in which the drug candidate product is effective at one time. Moreover, it is possible to construct a tissue having a function close to that of an actual biological tissue rather than a tissue-like structure in which cells are simply mixed at random. Furthermore, specific cells can be selectively isolated.

Claims (20)

The following formula (I)
[Where:
A ¹ is a lipid membrane-binding group that binds to the lipid membrane in the lipid membrane-containing material,
A ² is a lipid membrane binding inhibitor that inhibits the binding of the lipid membrane binding group to the lipid membrane;
A ³ is a photoreactive group,
A ⁴ is a hydrophilic group,
A ⁵ is a reactive group to the substrate to be modified,
A ⁶ is a scaffold site having at least three bonds,
L ¹ and L ² are linkers;
k ¹ and k ² are each independently 0 or 1]
The compound represented by the above, wherein the inhibition of binding by the lipid membrane binding inhibitory group is eliminated by a photoreaction, and the lipid membrane binding group can bind to the lipid membrane.   The compound according to claim 1, wherein the lipid membrane-containing material is selected from the group consisting of cells, organelles, vesicles, viruses, liposomes, micelles and quantum dots coated with lipids.   The compound according to claim 1 or 2, wherein the substrate to be modified is a substrate coated with at least one selected from the group consisting of bovine serum albumin, 3-aminopropyltriethoxysilane, and collagen. A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. 4. A group according to any one of claims 1 to 3, selected from the group, wherein adjacent carbon atoms in the _C7-22 alkyl group may be linked by 1 to 3 unsaturated bonds. Compound. A ³ is selected from the group consisting of 2-nitrobenzyl skeleton, coumarin-4-ylmethyl skeleton, phenylcarbonylmethyl skeleton, 7-nitroindolinocarbonyl skeleton, azobenzene skeleton, fulgide skeleton, spiropyran skeleton, spirooxazine skeleton and diarylethene skeleton The compound according to any one of claims 1 to 4, which is a divalent group having a skeleton. A ⁴ is selected from the group consisting of:
[Where:
Z is an oxyalkylene group having 2 to 4 carbon atoms, n represents the average number of added moles of the oxyalkylene group having 2 to 4 carbon atoms, and 2 ≦ n ≦ 500,
The left arrow indicates the connection to A ⁶ , the right arrow indicates the connection to A ⁵ when k ² is 0, and the connection to L ² when k ² is 1]
The compound of any one of Claims 1-5 represented by these. A ⁵ is the following substituent:
[Where:
The arrows indicate the connection to A ⁴ when k ² is 0, the connection to L ² when k ² is 1,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen and a C _1-10 alkoxy group]
The compound according to any one of claims 1 to 6, which is selected from the group consisting of: L ¹ and L ² are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _{1 10.} A compound according to any one of claims 1 to 7, optionally substituted with _-10 alkyl), O or S. A ⁶ is selected from the group consisting of:
[Where:
L ³ , L ⁴ and L ⁵ are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms among the carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), _optionally substituted with O or S,
k ³ , k ⁴ and k ⁵ are each independently 0 or 1,
Arrows to (A ¹ ), (A ³ ), and (A ⁴ ) indicate connections to A ¹ , A ^3, and A ⁴ , respectively]
The compound of any one of Claims 1-8 represented by these. The following formula (Ia):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group, and a C _7-22 alkyl C _6-14 aryl group. Selected from the group wherein adjacent carbon atoms in said C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
L ^{1 to} L ⁵ are each independently a C _6-14 arylene group or a C _1-10 alkylene group, wherein the carbon atom in the alkylene group is substituted with 1 to 5 oxo groups. Adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _{1 -10} alkyl), O or S may be substituted,
A ³ is a divalent group having a skeleton selected from the group consisting of a nitrobenzene skeleton, a coumarin skeleton, a hydroxyphenanthyl skeleton, a nitroindoline skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton. ,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to L ²
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound of any one of Claims 1-9 represented by these. The following formula (Ib):
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group, wherein adjacent carbon atoms in the C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
n represents the average number of moles added of oxyethylene groups, and 45 ≦ n ≦ 500,
p is 0-8,
q and s are each independently 0 to 7,
r is 0-10,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to the rest of the compound,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound of any one of Claims 1-10 represented by these. The following formula (Ic)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound of any one of Claims 1-11 represented by these. The following formula (II-a)
[Where:
A ¹ and A ² each independently comprise a C _7-22 alkyl group, a C _6-14 aryl group, a C _6-14 aryl C _7-22 alkyl group and a C _7-22 alkyl C _6-14 aryl group. Selected from the group, wherein adjacent carbon atoms in the C _7-22 alkyl group may be linked by 1 to 3 unsaturated bonds;
n represents the average number of moles added of oxyethylene groups, and 45 ≦ n ≦ 500,
p is 0-8,
q is each independently 0 to 7,
r is 0-10,
A ⁵ is the following substituent:
(Where
The arrow indicates the connection to the rest of the compound,
X is a halogen, and R ¹ , R ² and R ³ are each independently selected from the group consisting of a C _1-10 alkyl group, a halogen, and a C _1-10 alkoxy group)
Selected from the group consisting of]
The compound of any one of Claims 1-10 represented by these. The following formula (II-b)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound of Claim 13 represented by these. The following formula (Id)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
Or the following formula (II-c)
[Wherein n represents the average number of moles of oxyethylene group added and 45 ≦ n ≦ 500]
The compound of any one of Claims 1-10 represented by these.   A base material for immobilizing a lipid membrane-containing material obtained by reacting the compound according to any one of claims 1 to 15 with the base material to be modified. The group of formula (III) below:
[Where:
A ^{1 to} A ⁴ , A ⁶ , L ¹ , L ² , k ¹ and k ² are as defined in any one of claims 1 to 15;
A ⁷ is a divalent group that connects the other part of formula (III) and the substrate, or a reactive group that can be connected to the substrate by a non-covalent bond,
Arrow indicates covalent or non-covalent linkage to substrate]
A substrate for immobilizing a lipid membrane-containing material. A method for patterning a lipid membrane-containing material on a substrate, comprising the following steps:
(1) Irradiating light to a part that forms the pattern of the lipid membrane-containing material on the base material for immobilizing the lipid membrane-containing material according to claim 16 or 17, and the lipid membrane-binding group of the part A state capable of binding to a lipid membrane;
(2) The method comprising reacting the lipid membrane-containing material with the lipid membrane-binding group to form a pattern of the lipid membrane-containing material. The following steps:
(3) The substrate obtained in step (2) is washed to remove unreacted lipid membrane-containing material from the substrate;
(4) Using a lipid membrane containing material different from the lipid membrane containing material used in the previous step, repeating steps (1) to (3) to form a plurality of different lipid membrane containing patterns The method of claim 18 further comprising: A method for isolating a lipid membrane-containing material comprising the following steps:
(1) A sample containing a lipid membrane-containing material is applied to a substrate for immobilizing the lipid membrane-containing material according to claim 16 or 17;
(2) detecting the position of the lipid membrane-containing material on the substrate based on microscopic observation;
(3) irradiating the position with light so that the lipid membrane-bound group at the position can be bound to the lipid membrane, and immobilizing the lipid membrane-containing material on the substrate;
(4) The said method including wash | cleaning the base material obtained at step (3), and removing the substance which is not fix | immobilized in the base material from a base material.